Inactivating pathogens and producing highly immunogenic inactivated vaccines using a dual oxidation process

ABSTRACT

Provided are surprisingly effective methods for inactivating pathogens, and for producing highly immunogenic vaccine compositions containing an inactivated pathogen rendered noninfectious by exposure to a Fenton reagent, or by exposure to a Fenton reagent or a component thereof in combination with a methisazone reagent selected from the group consisting of methisazone, methisazone analogs, functional group(s)/substructure(s) of methisazone, and combinations thereof. The methods efficiently inactivate pathogens, while substantially retaining pathogen antigenicity and/or immunogenicity, and are suitable for inactivating pathogens, or for the preparation of vaccines for a wide variety of pathogens with genomes comprising RNA or DNA, including viruses and bacteria. Also provided are highly immunogenic inactivated vaccine compositions prepared by using any of the disclosed methods, and methods for eliciting an immune response in a subject by administering such vaccine compositions.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This work was supported at least in part by NIH Grant Nos. R44-AI079898and R01-A1098723, and the United States government therefore has certainrights.

FIELD OF THE INVENTION

Aspects of the present invention relate generally to methods forinactivating pathogens and producing highly immunogenic inactivatedvaccines against pathogens, relate in more particular aspects tosurprisingly effective methods for inactivating pathogens and producinghighly immunogenic inactivated vaccines against pathogens having eitherRNA or DNA genomes, including but not limited to viral and bacterialpathogens, using dual oxidation processes employing Fenton-typechemistry, and relate in even more particular aspects to using singleoxidation processes or the disclosed dual oxidation processes, incombination with a methisazone reagent (e.g., methisazone, methisazoneanalogs, or methisazone functional group(s)/substructure(s), orcombinations thereof), to provide substantial advantages over the use ofsingle or dual oxidation processes for viral, bacterial, fungal orparasite inactivation and vaccine production. Additional aspects relateto vaccine compositions (medicaments) containing a pathogen inactivatedusing the disclosed methods for producing highly immunogenic inactivatedvaccines, and to methods for eliciting an immune response in a subjectby administering such vaccine compositions.

BACKGROUND

Inactivated vaccines represent a critical component of the health caresystem for both human and veterinary fields of medicine. However, theprocess of inactivation (e.g., inactivation by formaldehyde,β-propiolactone (BPL), binary ethylenimine (BEI) inactivation, andhydrogen peroxide (H₂O₂)) can damage key antigenic epitopes of targetpathogens, leading to suboptimal in vitro and in vivo responses invaccines and reductions in in vivo vaccine efficacy.

Recent work (see, e.g., U.S. Pat. Nos. 8,124,397 and 8,716,000) hasshown that chemical oxidizing agents (e.g., hydrogen peroxide (H₂O₂)),while previously known and used in the art only for the ability todestroy and kill pathogens, could be used in methods to prepareimmunogenic inactivated viral vaccines. However, even such simplechemical oxidizing agents can give suboptimal results by damaging, tosome extent, key antigenic epitopes, and to circumvent this problem,there is yet a pronounced unmet need for better, broadly applicablemethods for efficiently inactivating pathogens (viral, bacterial,fungal, and parasitic) while optimally retaining immunogenicity.

Influenza, for example, commonly known as “the flu”, is an infectiousdisease caused by an influenza virus, RNA viruses that make up three ofthe five genera of the family Orthomyxoviridae. Influenza spreads aroundthe world in a yearly outbreak, resulting in about three to five millioncases of severe illness and about 250,000 to 500,000 deaths.

Dengue virus (DENV), for example, is the cause of dengue fever. It is amosquito-borne, positive-sense single stranded RNA virus of the familyFlaviviridae; genus Flavivirus. Five serotypes of the virus have beenfound, all of which can cause the full spectrum of disease. Its genomecodes for three structural proteins (capsid protein C, membrane proteinM, envelope protein E) and seven nonstructural proteins (NS1, NS2a,NS2b, NS3, NS4a, NS4b, NS5). It also includes short non-coding regionson both the 5′ and 3′ ends.

Chikungunya virus (CHIKV), for example, is a member of the alphavirusgenus, and Togaviridae family. It is an RNA virus with a positive-sensesingle-stranded genome of about 11.6kb. It is a member of the SemlikiForest virus complex and is closely related to Ross River virus,O′nyong'nyong virus, and Semliki Forest virus. Because it is transmittedby arthropods, namely mosquitoes, it can also be referred to as anarbovirus (arthropod-borne virus). In the United States, it isclassified as a category C priority pathogen, and work requiresbiosafety level III precautions. Symptoms include fever and joint pain,typically occurring two to twelve days after exposure. Other symptomsmay include headache, muscle pain, joint swelling, and a rash. Mostpeople are better within a week; however, occasionally the joint painmay last for months. The risk of death is around 1 in 1,000. The veryyoung, old, and those with other health problems are at risk of moresevere disease.

Campylobacter (Gram-negative bacteria), for example, represents a globalhuman pathogen and is responsible for up to 400-500 million cases ofbacterial gastroenteritis each year. The economic burden of thisbacterial disease is substantial, with annual US costs estimated at upto $5.6 billion. There is no commercial vaccine available for humanCampylobacter infections and development of a safe and effective vaccinerepresents an important unmet clinical need. The most frequentlyreported species in human diseases are C. jejuni (subspecies jejuni) andC. coli. Other species such as C. lari and C. upsaliensis have also beenisolated from patients with diarrhoeal disease, but are reported lessfrequently.

Listeria (e.g., Listeria monocytogenes; Gram-positive bacteria) is oneof the most virulent foodborne pathogens, with fatality rates due tofood-borne listeriosis reaching 20 to 30% in high-risk individuals.Responsible for an estimated 1,600 illnesses and 260 deaths in theUnited States (U.S.) annually, listeriosis ranks third in total numberof deaths among food borne bacterial pathogens, with fatality ratesexceeding even Salmonella and Clostridium botulinum. In the EuropeanUnion, rates of listeriosis have followed an upward trend that began in2008, causing 2,161 confirmed cases and 210 reported deaths in 2014, 16%more than in 2013. Similar to the U.S., listeriosis mortality rates arealso higher in the EU compared to other food-borne pathogens.

Shigella (e.g., Shigella dysenteriae; Gram-negative bacteria) is one ofthe leading bacterial causes of diarrhea worldwide, causing an estimated80-165 million cases annually. The number of deaths it causes each yearis estimated at between 74,000 and 600,000, and it is in the top fourpathogens that cause moderate-to-severe diarrhea in African and SouthAsian children. S. flexneri is the most frequently isolated speciesworldwide, and accounts for 60% of cases in the developing world; S.sonnei causes 77% of cases in the developed world, compared to only 15%of cases in the developing world; and S. dysenteriae is usually thecause of epidemics of dysentery, particularly in confined populationssuch as refugee camps.

The present disclosure satisfies these and other needs for bettervaccines.

SUMMARY OF THE INVENTION

Applicants herein disclose and demonstrate for the first time that useof a dual oxidation system, employing Fenton-type chemistry with, forexample, CuCl₂ and H₂O₂, as well as use of H₂O₂ with other transitionmetal/H₂O₂ combinations (Fenton reaction combinations), provided asignificant advantage in both inactivation and vaccine development overthe use of single oxidation approaches. Neither H₂O₂ nor CuCl₂ alone,for example, were able to maintain robust antigenicity while alsoensuring complete viral inactivation, both of which are criticalcomponents underlying successful inactivated vaccines. Surprisingly,using a combination of, for example, both CuCl₂ and H₂O₂, a broadvariety of antigenic and immunogenic vaccines were provided, forexample, for chikungunya virus (CHIKV, Family: Togaviridae, Genus:Alphavirus), dengue virus serotypes 1-4 (DENV 1-4) and yellow fevervirus (YFV), Family: Flaviviridae, Genus: Flavivirus), vaccinia virus(VV), Family: Poxviridae, Genus: Orthopoxvirus) or influenza virus(Family: Orthomyxoviridae, Genus: Influenzavirus) was developed.

Particular aspects, as described in more detail below, thus provide aneffective dual-oxidation method involving Fenton-type chemistry(oxidative reactions) using redox-active transition metals (e.g., Cu,Fe, Cs, etc.) in combination with hydrogen peroxide (H₂O₂) to formoxidative byproducts, leading to microbial inactivation withsurprisingly effective retention of immunogenicity.

In additional surprising aspects, the disclosed dual-oxidation methodsinvolving Fenton-type chemistry further comprise, as described in moredetail below, the use of methisazone, methisazone analogs, ormethisazone functional group(s)/substructure(s), providing even moreefficient microbial inactivation relative to dual-oxidation alone, andwith even more effective retention of immunogenicity relative todual-oxidation alone.

Further surprising aspects provide effective single-oxidation methodsinvolving hydrogen peroxide (H₂O₂) further comprising, as described inmore detail below, the use of methisazone, methisazone analogs, ormethisazone functional group(s)/substructure(s), providing for moreefficient microbial inactivation relative to H₂O₂ alone, and witheffective retention of immunogenicity.

Provided, for example, are methods for producing an immunogenic vaccinecomposition comprising an inactivated pathogen, the method comprising:contacting a pathogen with a Fenton reagent, comprising hydrogenperoxide in combination with a transition metal, in an amount and for atime-period sufficient for the agent to render the pathogennoninfectious while retaining pathogen immunogenicity. The methods mayfurther comprise verifying immunogenicity of the noninfectious pathogenusing pathogen-specific antibody, B cell or T cell immunoassays,agglutination assays, or other suitable assays. In the methods using aFenton reagent, the Fenton reagent comprises hydrogen peroxide incombination with at least one transition metal ion selected from, e.g.,Cu, Fe, Cs, etc., as recognized in the art. For the methods using aFenton reagent, a single transition metal, or a mixture of transitionmetals may be used in combination with hydrogen peroxide. The methodsare broadly applicable where the pathogen to be inactivated whileretaining immunogenicity is a pathogen having a genome comprising RNA orDNA, including but not limited to viruses, and bacteria, as disclosedherein. In particular aspects of the methods using a Fenton reagent thepathogen is a virus (e.g., Family Togaviridae, Flaviviridae, orOrthomyxoviridae) or bacterium (e.g., Campylobacter is C. coli or C.jejuni). In more particular aspects, the pathogen is a virus (e.g.,Togaviridae, Genus: Alphavirus), Family: Flaviviridae, Genus:Flavivirus) or Family: Orthomyxoviridae, Genus: Influenzavirus) orbacterium (e.g., Campylobacter). In particular aspects, the pathogen isa chikungunya virus (CHIKV, Family: Togaviridae, Genus: Alphavirus),dengue virus serotypes 1-4 and yellow fever virus (DENV 1-4, YFV,Family: Flaviviridae, Genus: Flavivirus) or influenza virus (Family:Orthomyxoviridae, Genus: Influenzavirus. In particular aspects, thepathogen is a bacterium such as, but not limited to Campylobacter (e.g.,C. coli or C. jejuni), Shigella spp, Listeria (e.g., Listeriamonocytogene), etc., as disclosed herein.

In the dual-oxidation or single-oxidation methods disclosed herein, thepathogen is preferably isolated or purified prior to contacting with theFenton reagent.

Also disclosed are methods for inactivating a pathogen, the methodcomprising: contacting a pathogen with hydrogen peroxide, or with aFenton reagent containing hydrogen peroxide in combination with atransition metal, and a methisazone reagent, in an amount and for atime-period sufficient to render the pathogen noninfectious.

The disclosed dual-oxidation methods disclosed herein for inactivatingpathogens, and for vaccine production by inactivating pathogens whileretaining immunogenicity, may comprise contacting the pathogen with theFenton reagent and a “methisazone reagent” such as methisazone, amethisazone analog(s), or one or more methisazone functionalgroup(s)/substructure(s), or combinations thereof. For example, thedual-oxidation methods described herein may comprise contacting thepathogen with the Fenton reagent and a compound having formula I:

wherein R₁ is independently H or lower alkyl (e.g., C1-C4 alkyl)optionally substituted with —OH; wherein R₂ is independently H, loweralkyl (e.g., C1-C2 alkyl) optionally substituted with —OH or with aryl;and wherein X is independently H or halogen (e.g., Cl, Br, I, F, etc.);and salts, including pharmaceutically acceptable salts thereof. Inparticular aspects, X and R₂ are H; and R₁ is independently H (isatin(β-thiosemicarbazone), -CH₃ (N-methyl-isatin β-thiosemicarbazone(methisazone)), or propyl (N-propyl-isatin β-thiosemicarbazone).Preferably, X and R₂ are H; and R₁ is -CH₃ (N-methyl-isatinβ-thiosemicarbazone (methisazone)). Preferably, methisazone is used:

Alternatively, or in addition, the dual-oxidation methods describedherein may comprise contacting the pathogen with the Fenton reagent andone or more compounds each having one of formulas II-V:

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH; and wherein X is independently H or halogen (e.g.,Cl, Br, I, F, etc.); and salts, including pharmaceutically acceptablesalts thereof;

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH; wherein X is independently H or halogen (e.g., Cl,Br, I, F, etc.); and wherein R₂ is independently H, lower alkyl (e.g.,C1-C2 alkyl) optionally substituted with —OH, or with aryl; and salts,including pharmaceutically acceptable salts thereof; and

wherein R₂ and R₃ are independently H, lower alkyl (e.g., C1-C2 alkyl)optionally substituted with —OH, or with aryl; and salts, includingpharmaceutically acceptable salts thereof; and combinations of suchcompounds each having one of the formulas (or each having one of theformulas I-V). Preferably: X of formula II is H, and R₁ of formula (II)is H (isatin), —CH₃ (N-methyl-isatin), or propyl (N-propyl-isatin); X,R₁ and R₂ of formula (III) are H (indole, 2,3-dione, 3-hydrazone); R₂and R₃ of formula (IV) are H (thiosemicarbazide); and R₂ and R₃ offormula (V) are H (semicarbazide). Preferably, contacting the pathogencomprises contacting the pathogen with the Fenton reagent,thiosemicarbazide and a compound having formula VI:

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl). Preferably, R₁ offormula VI is H (isatin), —CH₃ (N-methyl-isatin), or propyl(N-propyl-isatin). Preferably, R₁ of formula VI is H (isatin):

Also provided are immunogenic vaccine compositions having anoxidation-inactivated pathogen, produced by any of the methods disclosedherein. Preferably, the inactivated pathogen retains one or morepredominant antigenic epitopes of the biologically active pathogensuitable to elicit a pathogen-specific antibody, B cell or T cellresponse, or to reduce infection by the pathogen, or decrease symptomsthat result from infection by the pathogen. In the methods, the pathogengenome may comprise RNA or DNA. Additionally provided are methods foreliciting an immune response against a pathogen, the methods comprising:obtaining an immunogenic vaccine composition having anoxidation-inactivated pathogen, produced by any of the methods disclosedherein; and administering the immunogenic vaccine composition to asubject, thereby eliciting in the subject an immune response against thepathogen. In the methods, the pathogen genome may comprise RNA or DNA.

Further provided are methods for producing an immunogenic vaccinecomposition comprising an inactivated pathogen, the method comprising:contacting a pathogen with hydrogen peroxide in combination with amethisazone-type reagent selected from the group consisting ofmethisazone, a methisazone analog(s) (e.g., as described herein), amethisazone functional group/substructure (e.g., as described herein),and combinations thereof (e.g., as described herein), in an amount andfor a time-period sufficient to render the pathogen noninfectious whileretaining pathogen immunogenicity. Preferably, the methods furthercomprise verifying immunogenicity of the noninfectious pathogen usingpathogen-specific antibody, B cell or T cell immunoassays, agglutinationassays, or other suitable assays. In the methods, the pathogen genomemay comprise RNA or DNA.

Additionally provided are immunogenic vaccine compositions having anoxidation-inactivated pathogen, produced by the methods comprisingcontacting a pathogen with hydrogen peroxide in combination with amethisazone-type reagent.

Further provided are methods for eliciting an immune response against apathogen, the method comprising: obtaining an immunogenic vaccinecomposition having an inactivated pathogen, produced by the methodscomprising contacting a pathogen with hydrogen peroxide in combinationwith a methisazone-type reagent; and administering the immunogenicvaccine composition to a subject, thereby eliciting in the subject animmune response against the pathogen.

Further provided are methods for inactivating a pathogen, the methodcomprising: contacting a pathogen with a Fenton reagent, comprisinghydrogen peroxide in combination with a transition metal, and amethisazone reagent, in an amount and for a time-period sufficient forthe agent to render the pathogen noninfectious.

The methods are broadly applicable for producing highly immunogenicinactivated vaccines against pathogens having either RNA or DNA genomes,including but not limited to viral and bacterial pathogens.

The utility/efficacy/results are surprising and unexpected for at leastsix reasons.

First, prior to Applicants' U.S. Pat. Nos. 8,124,397 and 8,716,000(hereinafter '397″ and “'000” patents having claims encompassing use ofH₂O₂ alone in oxidative reactions for vaccine production), H₂O₂ wasregarded as a strong oxidant and thus H₂O₂ reactions were known and usedin the art only for the ability to destroy and kill pathogenseffectively, and there was no use, suggestion or reasonable expectationto use H₂O₂ oxidative reactions for immunogenic vaccine production assurprisingly disclosed in Applicants’ prior '397 and '000 patents.Likewise, prior to Applicants' present disclosure, and as discussed inmore detail below, Fenton-type oxidative reactions (H₂O₂+transitionmetal ions) were known in the art only for the ability to destroy andkill pathogens effectively, and there was no use, suggestion orreasonable expectation to use Fenton-type oxidative reactions forimmunogenic vaccine production as presently disclosed and claimed.Second, during the initial course of investigating the presentlydisclosed dual-oxidation approach using Fenton-type chemistry (H₂O₂ and+transition metal ions), it was discovered that virus inactivation usingFenton-type chemistry was viral protein concentration-dependent,completely unlike the case for H₂O₂ alone, which is not proteindependent (compare FIGS. 1A and 1B herein), indicating that afundamentally different mechanism was involved with Fenton-typechemistry-based pathogen inactivation (dual-oxidation system) comparedto H₂O₂ alone-based pathogen inactivation (single-oxidation system).Moreover, in the dual-oxidation system, the inactivation rate decreasedat higher viral protein concentrations, indicating that inclusion of theFenton-type chemistry may be targeting the viral protein antigens, whichcontraindicated use of Fenton-type chemistry-based pathogen inactivationin methods seeking to retain viral protein integrity/immunogenicity. Itwas, therefore surprising and unexpected that Fenton-typechemistry-based pathogen inactivation actually substantially improvedretention of viral protein integrity/immunogenicity, as disclosedherein.

Third, with respect to dual-oxidation methods further comprising the useof a methisazone reagent, there was no use or suggestion in the art touse a methisazone reagent (e.g., methisazone) in combination with aFenton reagent (e.g., with H₂O₂ and Cu), and thus no knowledge in theart about the potential effects, if any, of methisazone on Fenton-typechemistry in any context, including not in any vaccine preparationcontext. Applicants are in fact the first to disclose use of amethisazone reagent in combination with a Fenton reagent, as disclosedand claimed herein.

Fourth, as discussed in more detail below, methisazone was known in theart to combine with both nucleic acid and protein, and thus would becontraindicated for use in methods such as those disclosed herein, whichmethods are aimed at maximally retaining the integrity andimmunogenicity of pathogen protein epitopes, and particularly where therelevant pathogen protein epitopes are exposed on the pathogen surface,relative to the internally-sequestered nucleic acid of the pathogen.Moreover, the protein affinity of methisazone was particularlyconcerning given Applicants' initial finding, as discussed above, thatApplicants' dual-oxidation reactions were viral protein concentrationdependent (inactivation rate decreasing with increased viral proteinconcentration; FIG. 1B herein), thus contraindicating addition of yetanother agent that combines with or targets protein.

Fifth, methisazone was known in the art to complex/sequester transitionmetal ions, which would indicate to one of ordinary skill in thechemical arts the methisazone might competitively interfere with theFenton-type chemistry (H₂O₂+transition metal ions such as Cu), thuscontraindicating its use in combination with Fenton-type chemistry. Asdiscussed in more detail below, the metal ions are catalysts in theFenton-type oxidation reactions, and thus sequestration of suchcatalysts by methisazone reagents would be of particular concern.Surprisingly, however, methisazone reagents substantially increased boththe rate of Fenton-type chemistry-mediated pathogen inactivation, andthe retention of protein integrity/immunogenicity of the inactivatedpathogens.

Sixth, with respect to the disclosed methods for inactivating apathogen, no one in the art has previously inactivated a pathogen usingeither hydrogen peroxide plus a methisazone reagent, or using Fentonchemistry plus a methisazone reagent, and regardless of immunogenicityretention considerations, no one could have predicted increased rates ofpathogen inactivation relative to hydrogen peroxide alone, or Fentonchemistry alone.

For at least these six reasons, therefore, the results disclosed hereinwere surprising and unexpected, and could not have been predicted basedon either the prior art, or Applicants' own prior work with simplechemical oxidizing agents (e.g., H₂O₂) (U.S. Pat. Nos. 8,124,397 and8,716,000).

The advanced dual-oxidation methods were successfully applied to eightexemplary viral vaccine targets representing four unrelated virusfamilies (e.g., CHIKV, (Family: Togaviridae, Genus: Alphavirus), denguevirus serotypes 1-4 and yellow fever virus (DENV 1-4, YFV, Family:Flaviviridae, Genus: Flavivirus), vaccinia virus (VV), Family:Poxviridae, Genus: Orthopoxvirus) and influenza virus (Family:Orthomyxoviridae, Genus: Influenzavirus A)), and with respect to whichsimple oxidation (e.g., with H₂O₂ alone) was found to be suboptimal.

Additionally surprising, the advanced dual-oxidation methods were alsosuccessfully applied to bacterial vaccine targets (e.g., Campylobacter,Listeria, Shigella, etc.), in which simple oxidation (e.g., with H₂O₂alone) was found to be too destructive for vaccine development (e.g., inthe case of Campylobacter).

The disclosed dual-oxidation methods performed using Fenton-typechemistry (and optimally those methods described herein furthercomprising the use of a methisazone-type reagent selected from the groupconsisting of methisazone, methisazone analogs, methisazone functionalgroup(s)/substructure(s), and combinations thereof) provide for robustpathogen inactivation with maintained antigenic properties to providehighly effective vaccines, leading to enhanced immunologic responsesfollowing vaccination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show, according to particular aspects, that the kineticsof virus inactivation using the H₂O₂/CuCl₂ dual oxidation system isprotein concentration-dependent, whereas standard H₂O₂-based virusinactivation is protein concentration-independent. FIGS. 2A and 2B show,according to particular aspects, that standard H₂O₂-based inactivationdamages CHIKV-specific neutralizing epitopes, and fails to induceneutralizing responses in vivo following vaccination.

FIGS. 3A, 3B, and 3C show, according to particular aspects, that use ofthe disclosed dual oxidizing Fenton-type oxidation system providesefficient inactivation while improving the maintenance of CHIKV-specificneutralizing epitopes.

FIG. 4 shows, according to particular aspects, that CuCl₂/H₂O₂-CHIKVvaccination induces rapid neutralizing antibody responses.

FIGS. 5A and 5B show, according to particular aspects, thatCuCl₂/H₂O₂-CHIKV vaccination protects against CHIKV-associatedpathology.

FIGS. 6A and 6B show, according to particular aspects, that use of thedisclosed dual-oxidation approach with the yellow fever virus (YFV)demonstrates enhanced retention of antibody binding to neutralizingepitopes (antigenicity) and improved immunogenicity after vaccination.

FIG. 7 shows, according to particular aspects, that use of the discloseddual-oxidizing Fenton-type oxidation system demonstrates enhancedinactivation while maintaining dengue virus 3-specific neutralizingepitopes.

FIG. 8 shows, according to particular aspects, that use of the disclosedH₂O₂/CuCl₂ dual-oxidation system enhances in vivo immunogenicity to 3out of 4 DENV serogroups following immunization with a tetravalent DENVvaccine in rhesus macaques (RM).

FIG. 9 shows, according to particular aspects, that use of the disclosedH₂O₂/CuCl₂ dual-oxidation system enhances in vivo immunogenicity to 4out of 4 DENV serogroups following immunization with a tetravalent DENVvaccine in mice.

FIG. 10 shows, according to particular aspects, that the disclosedCuCl₂/H₂O₂-based virus inactivation maintains influenza hemagglutinationactivity significantly better than H₂O₂ alone.

FIGS. 11A and 11B show, according to particular aspects, that CuCl₂/H₂O₂inactivated influenza induces robust hemagglutination inhibition titersand protects against lethal challenge.

FIGS. 12A, 12B, and 12C show, according to particular aspects, acomparison of exemplary redox-active metals for the disclosed dualoxidation-based virus inactivation methods.

FIG. 13 shows, according to particular aspects, that combinations ofmetals can be used to achieve complete inactivation while maintaininggood antigenicity.

FIGS. 14A, 14B, and 14C show, according to particular aspects, use ofthe disclosed dual-oxidizing Fenton-type oxidation system for optimizedinactivation of Campylobacter for improved maintenance of bacterialmorphology.

FIG. 15 shows, according to particular aspects, exposure to an optimizedCuCl₂/H₂O₂ formula resulted in rapid inactivation of Campylobacter.

FIGS. 16A, 16B, and 16C show, according to particular aspects, thatCuCl₂/H₂O₂-C. coli is immunogenic and protects rhesus macaques (RM)against naturally acquired Campylobacter infection.

FIGS. 17A, 17B, and 17C show, according to particular aspects, thatmethisazone enhanced the rate of both single and dual oxidation-basedvirus inactivation.

FIGS. 18A, 18B and 18C show, according to particular aspects, thatmethisazone enhanced the rate of dual oxidation-based bacterialinactivation.

FIGS. 19A and 19B show, according to particular aspects, thatmethisazone enhanced inactivation rates while maintaining antigenicityduring dual oxidation-based viral inactivation.

FIGS. 20A, 20B, and 20C show, according to particular aspects, thatchemical analogs of methisazone, or methisazone functionalgroups/substructures, enhanced inactivation and maintenance ofantigenicity during dual oxidation-based viral inactivation.

FIG. 21 shows, according to particular aspects, that increasing levelsof methisazone relative to the transition metal component of the dualoxidation system improved the antigenicity and inactivation profile ofthe dual oxidation system.

DETAILED DESCRIPTION OF THE INVENTION

While inactivated vaccines represent a critical component of the healthcare system for both human and veterinary fields of medicine, the priorart processes of inactivation damage key antigenic epitopes of targetpathogens (e.g., viral and bacterial), leading to suboptimal responsesin vaccines and reductions in vaccine efficacy.

Particular aspects of the present invention circumvent this problem byproviding a new dual-oxidation approach involving Fenton-type chemistry.Fenton-type oxidative reactions require the use of redox-activetransition metals (e.g., Cu, Fe, Cs, etc.) in combination with hydrogenperoxide (H₂O₂) to form oxidative byproducts, leading to microbialinactivation.

The disclosed advanced Fenton-type dual-oxidation process wassuccessfully applied to pathogens having either RNA or DNA genomes,including three exemplary bacteria (both Gram-positive and Gram negativeexamples, all with DNA genomes) including Campylobacter (e.g., C. colior C. jejuni), Shigella spp, and Listeria (e.g., Listeriamonocytogenes), and eight viruses (7 RNA genome viruses and 1 DNA genomevirus) in four unrelated virus families as vaccine targets (e.g.,chikungunya virus (CHIKV, Family: Togaviridae, Genus: Alphavirus),dengue virus serotypes 1-4 (DENV1, DENV2, DENV3, DENV4) and yellow fevervirus (YFV), Family: Flaviviridae, Genus: Flavivirus), vaccinia virus(VV), Family: Poxviridae, Genus: Orthopoxvirus) and influenza virus(Family: Orthomyxoviridae, Genus: Influenzavirus A)) in which simpleoxidation (e.g., hydrogen peroxide (H₂O₂) alone) was found to besuboptimal. For CHIKV, DENV and YFV, in vitro antigenicity was assessedthrough virus-specific ELISA tests based on monoclonal antibodiesdirected at sensitive neutralizing epitopes. Antigenicity for influenzawas assessed through hemagglutination activity (HA), a directmeasurement of viral protein function. In vivo enhancement of vaccineantigens was assessed through functional humoral immune assays, such asneutralizing antibody titers (CHIKV, DENV and YFV) or hemagglutinationinhibition (HAI, influenza) responses following vaccination.

The disclosed dual oxidation-based inactivation conditions weresuccessfully demonstrated to enhance maintenance of in vitroantigenicity when compared, for example, to H₂O₂ alone, and for CHIKV,DENY, YFV and influenza, the dual-oxidation based inactivation approachdemonstrated high antigenicity as well as complete virus inactivation.When these vaccines were tested in vivo, they provided antiviral immuneresponses equivalent or superior to that achieved through standardH₂O₂-based inactivation conditions, and in some cases equivalent to thatseen with live virus. The disclosed dual-oxidation performed usingFenton-type chemistry thus provides robust pathogen inactivation withmaintained antigenic properties, and enhances immunologic responsesfollowing vaccination.

Fenton-Type Reactions

Fenton-type chemical reactions are generally described (e.g., byBarbusiński, K., Fenton Reaction—Controversy concerning the chemistry.Ecological Chemistry and Engineering, 2009. 16(3): p. 347-358;incorporated herein in its entirety for its teachings related toFenton-type reactants and reactions) using the following chemicalequation:

M^(n+)+H₂O₂→M^((n+1)+)+HO⁻+HO⋅  (eq. 1)

In equation 1, M is a transition metal that can interact with H₂O₂. Thisreaction leads to the decomposition of H₂O₂, resulting in the productionof a hydroxyl ion (HO⁻) and the highly reactive hydroxyl radical (HO⋅).Note that certain transition metals, such as Fe and Cu, are consideredparticularly redox active, and most efficient in promoting thisreaction. In the complete reaction, the metal ion is returned to itsoriginal oxidation state through an additional reaction with H₂O₂,making the metal ion a true catalyst (Id). As an example, the overallreaction with Cu²⁺ can be written as follows:

Cu²⁺+H₂O₂→Cu⁺+HO₂⋅+H⁺  (eq. 2)

Cu⁺+H₂O₂→Cu²⁺+HO⁻+HO⋅  (eq. 3)

In equation 2, H₂O₂ acts as a reducing agent, reducing Cu²⁺ to Cu⁺. Inequation 3, the Cu⁻ in turn reduces H₂O₂ leading to the production ofthe reactive hydroxyl radical and return to the Cu²⁺ oxidation state,allowing subsequent rounds of catalysis. As described, there may beadditional side reactions that occur during Fenton-type reactions (Id).

Prior Art Use of Fenton-Type Oxidation was Only as a Broad-BasedSterilization and Pathogen Decontamination System

As mentioned above, similar to case for the standalone disinfectant usesof H₂O₂ prior to Applicants' U.S. Pat. Nos. 8,124,397 and 8,716,000,prior to the present disclosure, Fenton-type reactions were known in theart only for inactivation/sterilization of microbial pathogens (e.g.,see Sagripanti, J.L., L.B. Routson, and C.D. Lytle, Virus inactivationby copper or iron ions alone and in the presence of peroxide. ApplEnviron Microbiol, 1993. 59(12): p. 4374-6; Nieto-Juarez, J. I., et al.,Inactivation of MS2 coliphage in Fenton and Fenton-like systems: role oftransition metals, hydrogen peroxide and sunlight. Environ Sci Technol,2010. 44(9): p. 3351-6).

For example, Fenton-type oxidation has been recognized by multiplegroups as a potent antimicrobial platform. FDA researchers firstdetailed the systematic study of Fenton-type reactions as anantimicrobial approach, specifically aimed towards use in thesterilization of medical devices (Sagripanti, J. L., Metal-basedformulations with high microbicidal activity. Appl Environ Microbiol,1992. 58(9): p. 3157-62). Using the Junin virus (ssRNA, Genus:Arenavirus) as a model pathogen, rapid inactivation was observed withboth Fe³⁺ and Cu²⁺ as catalysts in the redox reaction (eq. 1), workingas well as standard sterilization approaches (2% glutaraldehyde)following optimization. As noted by the author at the time, the use ofeither of these metals was particularly attractive for medical purposes,given that normal human serum contains relatively high amounts of bothFe and Cu. For instance, total serum Cu levels in normal subjects rangesfrom 700-1500 μg/L (11-24 μM) (McClatchey, K. D., Clinical laboratorymedicine. 2nd ed. 2002, Philadelphia: Lippincott Wiliams & Wilkins. xiv,p. 452), while Fe levels range from 500-1700 μg/L (9-30 μM) (LippincottWilliams & Wilkins., Nursing. Deciphering diagnostic tests. Nursing.2008, Philadelphia, Pa.: Wolters Kluwer/Lippincott Williams & Wilkins.vii, p. 13). This same research group continued to expand on theCu-based Fenton reaction, demonstrating antimicrobial activity againstmultiple viral targets such as ϕX174 bacteriophage (ssDNA), T7bacteriophage (dsDNA), herpes simplex virus (HSV, dsDNA) and ϕ6bacteriophage (dsRNA) (Sagripanti, J. L., L. B. Routson, and C. D.Lytle, Virus inactivation by copper or iron ions alone and in thepresence of peroxide. Appl Environ Microbiol, 1993. 59(12): p. 4374-6).Additional studies with HSV using the H₂O₂/Cu²⁺ system (0.01% H₂O₂, 16μM Cu²⁺) confirmed rapid inactivation and suggested that directoxidation of nucleic acid underpins the viral inactivation (Sagripanti,J. L., et al., Mechanism of copper-mediated inactivation of herpessimplex virus. Antimicrob Agents Chemother, 1997. 41(4): p. 812-7), withsupporting studies demonstrating the high affinity of Cu²⁺ for nucleicacids (Sagripanti, J. L., P. L. Goering, and A. Lamanna, Interaction ofcopper with DNA and antagonism by other metals. Toxicol Appl Pharmacol,1991. 110(3): p. 477-85) and the ability of H₂O₂/Cu²⁺ systems to inducestrand breaks in nucleic acids (Toyokuni, S. and J. L. Sagripanti,Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strandbreaks mediated by copper and iron, in Free Radic Biol Med. 1996: UnitedStates. p. 859-64). Several other groups have also demonstrated thepathogen inactivation potential of H₂O₂/Cu²⁺ systems. Nieto-Juarez, et.al., demonstrated rapid inactivation of MS2 bacteriophage (ssRNA) using50 μM H₂O₂ (0.00017%) and 1 μM Cu²⁺, with the authors suggesting itspotential for wastewater decontamination (Nieto-Juarez, J. I., et al.,Inactivation of MS2 coliphage in Fenton and Fenton-like systems: role oftransition metals, hydrogen peroxide and sunlight. Environ Sci Technol,2010. 44(9): p. 3351-6) (see also Nguyen, T. T., et al., Microbialinactivation by cupric ion in combination with H ₂ O ₂ : role ofreactive oxidants. Environ Sci Technol, 2013. 47(23): p. 13661-7).

In total, these prior art studies were strictly in the context ofdecontamination, and merely demonstrate that the H₂O₂/Cu²⁺ system wasknown to be able to efficiently kill/sterilize model pathogens.

Simple Oxidation With H₂O₂ Limited Vaccine Immunogenicity With CertainPathogen Targets

Applicants have previously shown (e.g., U.S. Pat. Nos. 8,124,397 and8,716,000) that sole use of H₂O₂ as a simple oxidation agent providessuitable inactivation agent for various vaccine candidates.

However, during continued development of oxidizing with H₂O₂ alone,instances with certain pathogens in which antigenicity andimmunogenicity were reduced during the inactivation process wereencountered. For example, during recent early-stage development of achikungunya virus (CHIKV) vaccine candidate, we found as presentedherein under working Example 1, that treatment with 3% H₂O₂ understandard conditions destroyed neutralizing epitopes and led to a nearlycomplete loss of antigenicity, as judged through in vitro potencytesting using envelope-specific MAbs (FIG. 2A). This loss of measuredantigenicity had significant implications for in vivo immunogenicitysince H₂O₂-inactivated CHIKV-immunized animals were unable to mountmeasurable neutralizing antiviral antibody responses (FIG. 2B).

Dual Oxidation-Based Microbial Inactivation was Found by Applicants toHave a Fundamentally Different Mechanism Compared With Simple OxidationWith H₂O₂ Alone, Thereby Initially Discouraging the Potential Use ofDual Oxidation-Based Microbial Inactivation for the Development ofAdvanced Efficacious Vaccine Antigens

While Fenton-type reactions have only been used in the prior art forkilling pathogens, and have not been used or suggested for use in thedevelopment of vaccines, Applicants nonetheless tested, as shown hereinunder working Example 2, such reactions for the potential to inactivatemicrobial pathogens for purpose of vaccine production. The initialinactivation data was surprising and unexpected, because in contrast toH₂O₂, it was found that the total protein concentration of the solutionduring the inactivation procedure impacts H₂O₂/CuCl₂ dual-oxidationinactivation kinetics. Protein concentration had been previously shownto have no impact on viral inactivation using Applicants' standard H₂O₂approach. As shown in FIGS. 1A and 1B for DENV2, using the dualoxidation approach, protein concentration had a substantial impact inviral inactivation kinetics, with higher protein levels leading toslower inactivation of the virus.

The unexpected dependence on total protein concentration of the solutionduring the dual inactivation indicated that a fundamentally differentmechanism was involved compared to H₂O₂ alone as in Applicants' priorsimple oxidation based methods (e.g., with H₂O₂ alone) (e.g., U.S. Pat.Nos. 8,124,397 and 8,716,000), and thus the efficacy/use of a dualoxidation-based inactivation procedure for effective vaccine productionwas entirely questionable and unpredictable.

Applicants, despite the discovery of a different, proteinconcentration-dependent mechanism, nonetheless performed additionalexperiments discussed herein and included in the working examples below,to show that Fenton-type dual oxidation reactions can surprisingly beused to effectively inactivate microbial pathogens, and provide forhighly immunogenic and effective vaccines.

Dual Oxidation-Based Inactivation in the Development of Advanced VaccineAntigens

The Fenton-type oxidation (e.g., the H₂O₂/Cu²⁺ system) has not been usedor suggested for use in the art for the development of vaccines. DespiteApplicants' discovery that a fundamentally different mechanism wasinvolved (i.e., protein concentration dependence), Applicantsnonetheless explored this system's utility in the development of avaccine candidate against CHIKV, as this target had demonstrated poorimmunogenicity with no induction of neutralizing antibodies using astandard H₂O₂ inactivation approach (FIGS. 2A and 2B).

Each component of the system alone (H₂O₂ or CuCl₂, a source of Cu²⁺ions) was first assessed in terms of their respective ability to fullyinactivate virus while maintaining appropriate antigenicity.Antigenicity is defined by the ability to measure intact proteinepitopes on the virus surface using monoclonal antibodies that bindspecific virus neutralizing epitopes. Alternatively, structuralantigenicity can also be defined by physiologic protein function/bindingassays, such as those used to measure hemagglutination activity ofinfluenza virus. The antigenicity results based on monoclonal antibodybinding to CHIKV are shown herein under working Example 3.

Increasing concentrations of either decontamination reagent (FIGS. 3Aand 3B) led to enhanced inactivation, but at the expense ofsignificantly decreased antigenicity due to damage of neutralizingepitopes.

Surprisingly, by contrast, using the combined H₂O₂/CuCl₂ system, anoptimal inactivation condition was identified that fully maintainedantigenicity while leading to complete viral inactivation (FIG. 3C).

CuCl₂/H₂O₂-CHIKV vaccination generated rapid and robust neutralizingantibody titers, and demonstrated full protection against arthriticdisease

To assess the immunogenicity of the H₂O₂/CuCl₂-treated CHIKV candidate,vaccine antigen was formulated with alum adjuvant and used to immunizemice at several dose levels (10 or 40 μg per animal). As shown hereinunder working Example 4, CuCl₂/H₂O₂-CHIKV vaccination generated rapidand robust neutralizing antibody titers (FIG. 4), and demonstrated fullprotection against arthritic disease (FIG. 5).

H₂O₂/CuCl₂-Based Oxidation was Successfully Used in the Development ofan Inactivated YFV Vaccine

Based on the encouraging results demonstrated with CHIKV, a modelalphavirus, the utility of the system for flaviviruses such as YFV wasexplored.

As shown herein under working Example 5, preliminary analysis suggestedthat a concentration of 0.002% H₂O₂ and 1 μM CuCl₂ represented afunctional balance between antigenicity and rapid virus inactivation(FIG. 6A). Using a further optimized condition of 0.10% H₂O₂ and 1 μMCuCl₂ (to ensure full inactivation) vaccine material was produced forYFV and used to immunize adult BALB/c mice. Following vaccination, allanimals demonstrated measurable neutralizing titers with an averageneutralizing titer of 240, compared to a neutralizing titer of less than40 for animals immunized with YFV vaccine prepared using H₂O₂ alone(FIG. 6B). These differences in immunogenicity after vaccination couldbe anticipated based on the severe damage to neutralizing epitopes(i.e., antigenicity) observed when YFV was treated with 3% H₂O₂ for 20hours. FIGS. 6A and 6B show that H₂O₂/CuCl₂-based oxidation wassuccessfully used in the development of an inactivated YFV vaccine.

H₂O₂/CuCl₂-Based Oxidation was Successfully Used in the Development ofan Inactivated DENV Vaccine

Based on the encouraging results demonstrated with YFV, another modelflavivirus, dengue 3 (DENV3) was tested in the H₂O₂/CuCl₂ system.

As shown herein under working Example 6, as with YFV, initial testsindicated that a concentration of 0.002% H₂O₂ and 1 μM CuCl₂ representedan optimal approach for maintaining high antigenicity while alsoproviding complete virus inactivation (FIG. 7). Using these preliminaryH₂O₂/CuCl₂ inactivation conditions, vaccine lots of each DENV serotypewere produced, formulated into a tetravalent dengue vaccine adjuvantedwith 0.10% aluminum hydroxide, and used to immunize adult rhesusmacaques. Following a single booster immunization, all monkeysseroconverted (NT₅₀≥10), with the H₂O₂/CuCl₂ inactivation approachdemonstrating an improvement in neutralizing antibody responses for 3out of 4 dengue virus serotypes and an average 8-fold increase ingeometric mean titers when compared to inactivation with H₂O₂ alone(FIG. 8). There was a small difference in antigen dose (1 μg/serotypevs. 2 μg/serotype) in these studies and so the experiment was repeatedin mice that were vaccinated with the same dose of tetravalent denguevaccine antigen (FIG. 9).

In these experiments, the dual oxidation approach of H₂O₂/CuCl₂inactivation was more immunogenic than 3% H₂O₂ for all 4 dengue virusserotypes and resulted in an 8-fold to >800-fold increase inneutralizing antibody titers.

CuCl₂/H₂O₂-based oxidation demonstrated improved antigenicity withinfluenza virus

Given the positive results observed across two virus families(Togaviridae and Flaviviridae), an additional virus family was chosen totest using this new inactivation platform.

As shown herein under working Example 7, inactivation of Influenza Avirus (family Orthomyxoviridae) was tested using a standard 3% H₂O₂approach, ultraviolet inactivation, or the optimized CuCl₂/H₂O₂ system(0.002% H₂O₂ and 1 μM CuCl₂). To assess antigenicity, a hemagglutinationactivity (HA) titration assay was used. Influenza viruses naturallyagglutinate red blood cells, and maintenance of this activity throughoutinactivation is considered key to the immunogenicity of the finalvaccine product. As shown in FIG. 10, Applicants' CuCl₂/H₂O₂ systemmaintained HA titers similar to that observed for live, untreatedantigen. By comparison, UV inactivation reduced HA activity to anegligible level. The in vivo consequence of this HA destruction can beseen in FIG. 11, with the CuCl₂/H₂O₂ inducing robust protective serumantibody hemagglutinin inhibition (HAI) titers, while UV-treated antigeninduced no functional antibodies in mice and minimal protection againstlethal challenge.

Multiple Transition Metals can be Used in the Dual-Oxidation Approach toVaccine Antigen Development

Cu²⁺ (in the form of CuCl₂) was the initial metal tested in thedual-oxidation vaccine antigen development studies described for CHIKV,DENY, YFV and influenza virus. However, as described above, other metalsalso have the potential to function in a similar manner.

As shown herein under working Example 8, using DENV3 as a model virus,inactivation studies consisting of CuCl₂ (Cu²⁺), FeCl₃ (Fe³⁺) or CsCl(Cs⁺) and dilutions of H₂O₂ were tested for their potential in thedevelopment of vaccine antigen.

As shown in FIGS. 12A-12C, all three metals provided conditions thatmaintained high levels of antigenicity while demonstrating completevirus inactivation.

Combinations of Transition Metals Demonstrate Synergy in theDual-Oxidation Vaccine System

As shown above in FIG. 11 and working Example 8, different metals can beused in combination to enhance H₂O₂ inactivation of viruses.

As shown herein under working example 9, to investigate potentialsynergistic effects, DENV3 model virus was inactivated with combinationsof CuCl₂ (Cu²⁺) and FeCl₃ (Fe³⁺) at a set amount of H₂O₂ (0.01%). Anumber of CuCl₂/FeCl₃ conditions provided full inactivation whilemaintaining good antigenicity, demonstrating that using multiple metalsin the same inactivation condition is feasible (FIG. 13). Indeed, atCuCl₂ concentrations of 0.05 μM and 0.10 μM, increasing FeCl₃concentrations enhanced antigenicity, indicating synergy with these twometals.

Dual Oxidation was Used to Provide Optimized Inactivation ofCampylobacter for Improved Maintenance of Bacterial Morphology

As shown herein under working Example 10, Campylobacter are smallcorkscrew-shaped bacteria that are typically ˜0.2 μm in diameter and˜2-8 μm in length (FIG. 14A).

Following inactivation with a standard 3% H₂O₂ solution for 5 hours atroom temperature, the bacteria were substantially damaged with clearchanges in morphology, including loss of gross cellular structure andsubstantial clumping (FIG. 14B).

However, upon optimization of a dual-oxidation approach using 0.01% H₂O₂and 2 μM CuCl₂, Applicants surprisingly found that dual oxidation couldcompletely inactivate Campylobacter coli (C. coli) while maintainingexcellent bacterial morphology throughout the treatment period withmicrobes that remained indistinguishable from the untreated controls(FIG. 14C).

In addition to retained structure, a critical parameter for preparing aninactivated whole-cell vaccine is to ensure complete microbeinactivation. Using the optimal conditions described above, inactivationkinetic studies were performed. As shown in FIG. 15, C. colidemonstrated rapid inactivation, with a decay rate half-life of(T_(1/2)) of ˜15 minutes. These kinetics indicate >20 logs ofinactivation during the full 20-hr inactivation period. Based on thebacterial titers in the pilot manufacturing lots (˜10⁹ CFU/mL) thislevel of inactivation provides a high safety margin during themanufacturing process (up to 100 million-fold theoretical excessinactivation) while still maintaining overall bacterial structure (FIG.14C).

CuCl₂/H₂O₂-C. coli vaccination provided protective immunity in rhesusmacaques

As shown herein under working Example 11, Applicants determined vaccineefficacy in 60 CuCl₂/H₂O₂-C. coli-immunized rhesus macaques from twooutdoor sheltered housing groups, and then monitored the animals forCampylobacter culture-confirmed enteric disease.

For this study, animals were vaccinated intramuscularly with theCuCl₂/H₂O₂-C. coli vaccine candidate (inactivated using 0.01% H₂O₂ and 2μM CuCl₂), with a booster dose administered 6-months later. Vaccinatedgroups were selected based on prior disease history, with preferencegiven to groups that had historically high incidence rates ofCampylobacter infection. This approach provided increased robustness inevaluating protective efficacy. All adults/juveniles (n=59) received a40-μg alum-adjuvanted dose, with 2 small infants (<2 Kg body weight)receiving a half-dose (20-μg). According to protocol, any animaldiagnosed with Campylobacter-associated diarrhea during the first 14days after vaccination would be excluded since vaccine-mediatedprotection would be unlikely to occur during this early period. Oneadult animal was excluded from the study due to Campylobacter-associateddiarrhea on the day after vaccination. Serum samples were collected fromall remaining vaccinated animals (n=59) at day 0 and at 6 months afterprimary vaccination at which time the animals received a booster dose ofvaccine.

Following primary vaccination, the Applicants observed a significantincrease in Campylobacter-specific serum antibody titers (FIG. 16A,P<0.001) in addition to protection against Campylobacter-associateddiarrheal disease in comparison with prior years within the same sheltergroup (FIG. 16B, P=0.038) or in comparison with other shelter groupsduring the 2015 Campylobacter season (FIG. 16C, P=0.020). The health ofNHP are monitored daily and cases of diarrheal disease are documented ina searchable central database. Diarrhea incidence was monitored in thevaccinated cohort and compared to approximately 1,000 unvaccinatedcontrol animals in other similar shelter groups. Fecal samples werecollected from any animal experiencing a diarrheal episode and testedfor C. coli, C. jejuni, and Shigella spp. since these represent the mainenteric pathogens associated with diarrhea among the animals.

Interim analysis at 6 months after primary vaccination demonstrated nocases of C. coli or C. jejuni-associated diarrhea in the vaccinatedgroup versus 76 cases of Campylobacter-associated diarrhea among theunvaccinated animals, representing a statistically significantprotective effect against Campylobacter culture-positive diarrhealdisease (P=0.035) after a single vaccination.

Since nearly all human vaccines require at least two doses for optimalprotective efficacy and the durability of immunological memory is oftenimproved following booster vaccination, the Applicants followed theconservative approach of administering a booster vaccination at the 6month time point and then continued to monitor the incidence ofdiarrheal disease among the NHP. At 250 days after primary vaccination,more cases of Campylobacter-associated enteric disease had continued toaccrue among the unvaccinated population (reaching 8.7% or a total of 92animals) whereas none of the animals (0/59) in the vaccinated cohortshowed signs of disease and the statistical significance between the twogroups increased to P=0.020.

Methisazone Reagents

As disclosed and discussed in detail above, oxidizing transition metals(e.g., Cu²⁺, Fe³⁺, etc.) can be used in conjunction with ourperoxide-based vaccine development platform to enhance virusinactivation while limiting antigenic damage. However, for somepathogens it was noted that antigenic degradation can occur even whenusing this advanced dual-oxidation approach. To further improve vaccinedevelopment, additional compounds were searched/screened for the abilityto interact synergistically with our disclosed dual-oxidation-basedinactivation approach to increase the rate of inactivation while furtherreducing damage to immunogenic protein antigens. Through this search,methisazone (N-methylisatin β-thiosemicarbazone, CAS 1910-68-5;C₁₀H₁₀N₄OS; MWt 234.3 Da; Synonyms: metisazone; Marboran; Marborane;33T57; M-IBT; 1-methylisatin 3-thiosemicarbazide; N-methylisatinβ-thiosemicarbazone) was identified by Applicants. Methisazone is one ofa series of antiviral drugs developed by the Wellcome Foundation in the1950s (Thompson R L, et al., J Immunol. 1953;70:229-34; Bauer D. J., BrJ Exp Pathol. 1955;36:105-14). Based on small animal efficacy studieswith orthopoxviruses, methisazone was developed into the commercialproduct, Marboran®, and tested in several clinical trials including boththe treatment of vaccinia complications, as well as prophylaxis andtreatment for smallpox (Bauer D J., Ann N Y Acad Sci. 1965; 130:110-7).

According to Bauer (Id), early case reports for the use of methisazonein the treatment of vaccinia complications (eczema vaccinatum andvaccinia gangrenosa) indicate it may have been effective, but the lackof controls and concomitant use of antivaccinial gamma globulin (in somecases) makes it challenging to confirm efficacy. Nevertheless, the lackof serious adverse events is encouraging. Mean initial doses were 152mg/kg, with a total average dose of 809 mg/kg given over 3.75 days. Foran estimated human subject weight of 70 kg, this would translate into˜10 gr per dose, and ˜60 gr per treatment course. Bauer mentions thatmethisazone was used prophylactically prior to vaccinia vaccination, andwas reported to reduce complications (Id).

Thus, historical in vivo data demonstrates that methisazone is safe andeven trace amounts of this compound will not be an issue in new vaccineand drug products.

Some of the most impressive data for methisazone relates to smallpoxprophylaxis as reported during an outbreak in Madras, India (Bauer D Jet al., Lancet, 1963;2:494-6). Of the close contacts receivingmethisazone, only 3/1101 (0.27%) developed mild smallpox (no deaths),while 78/1126 (6.9%) developed smallpox, with 12 deaths. When focusingon only non-vaccinated subjects, 2/102 methisazone-treated subjectscontracted smallpox (2%) while 28/100 (28%) of untreated controlscontracted smallpox, with 11 deaths. Dosages were altered somewhatthroughout the trial and consisted of either (1) 1.5 gr by mouth twicedaily after meals for 4 days (12 gr total); (2) 3 gr by mouth twicedaily after meals for 4 days (24 gr total); (3) two doses of 3 gr bymouth within a 12 hr period (6 gr total). Methisazone, in combinationwith CuSO₄, has been described for the decontamination of viruses (Fox MP, et al., Ann N Y Acad Sci. 1977; 284:533-43; Logan J C, et al., J GenVirol. 1975; 28:271-83), but not for vaccine production, and has neverbeen used in conjunction with H₂O₂.

Fenton-Type Chemistry Plus Methisazone Reagents

Surprisingly, Applicants discovered that methisazone reagents, asdescribed herein, interact synergistically with the presently discloseddual-oxidation-based inactivation approach to substantially increase therate of inactivation while further reducing damage to immunogenicprotein antigens.

In additional aspects, therefore, the disclosed dual-oxidation methodsinvolving Fenton-type chemistry further comprise, as described in moredetail below in the working Examples, the use of methisazone,methisazone analogs, or methisazone functional group(s)/substructure(s),providing even more efficient microbial inactivation relative todual-oxidation alone, and with even more effective retention ofimmunogenicity relative to dual-oxidation alone.

The exact mode of action for methisazone in the disclosed methods isunclear, though studies have shown that methisazone can complex withcopper, and this complex has the capacity to bind both nucleic acid(Mikelens P E, et al., Biochem Pharmacol. 1976; 25:821-7) and protein(Rohde W, et al., J Inorg Biochem. 1979; 10:183-94). To explainApplicants' results, without being bound by mechanism, Applicantshypothesized that the methisazone-copper complex might preferentiallybind nucleic acid of the whole pathogen, and once bound, H₂O₂ may theninteract with the Cu²⁺ of the methisazone-copper complex in a classicFenton-type reaction to release highly active hydroxyl radicals in theproximity of the bound nucleic acid (e.g., a nucleic acid-focusedoxidation). This release of oxidative radicals may then lead tosubstantial, but localized, damage of the nucleic acid and inactivationof the pathogen. Applicants speculated, therefore, that lower amounts ofH₂O₂ than would typically be needed to inactivate pathogens could beused, thus limiting off-site/collateral damage to protein epitopes.Additionally, or alternatively, isatin β-thiosemicarbazone compoundshave also been shown to directly bind nucleic acid (Pakravan &Masoudian, Iran J Pharm Res. 2015; 14:111-23), suggesting that thisclass of compounds alone may be able to open up nucleic acidmacromolecules (e.g., by intercalation, and/or minor groove binding).Applicant speculated that if this was true, it may allow for greateraccess of oxidizing agents to the nucleic acid target to enhanceoxidation-based virus inactivation.

Methisazone Enhanced the Rate of Both Single and Dual Oxidation-BasedVirus Inactivation

As shown herein under working Example 12, Applicants determined thatmethisazone enhanced the rate of both single and dual oxidation-basedvirus inactivation. As shown in FIGS. 17A-C, the addition of methisazonewas able to substantially increase the rate of dual-oxidation-basedinactivation for vaccinia virus (VV, DNA genome) as well as dengue virusserotype 4 (DENV4, RNA genome) and chikungunya virus (CHIKV, RNAgenome).

Further, while methisazone alone had a minimal impact on virusinactivation (FIGS. 17B & 17C), methisazone and H₂O₂ together (even inthe absence of copper) demonstrated a synergistic enhancement for virusinactivation. Further surprising aspects, therefore, provide effectivesingle-oxidation methods involving hydrogen peroxide (H₂O₂) furthercomprising, as described in more detail below, the use of methisazone,methisazone analogs, or methisazone functional group(s)/substructure(s),providing for more efficient microbial inactivation relative to H₂O₂alone, and with effective retention of immunogenicity.

Methisazone Enhanced the Rate of Dual Oxidation-Based BacterialInactivation

As shown herein under working Example 13, Applicants determined thatmethisazone enhanced the rate of dual oxidation-based bacterialinactivation.

The results of working Example 12 were extended to DNA-encoded bacteria(FIGS. 18A-C) where again the addition of methisazone to thedual-oxidation approach (e.g., H₂O₂/CuCl₂) substantially enhancedinactivation rates for Campylobacter coli (an exemplary gram-negativebacteria), Listeria monocytogenes (an exemplary gram-positive bacteria)and Shigella dysenteriae (an exemplary gram-negative bacteria).

Methisazone Enhanced Inactivation Rates While Maintaining AntigenicityDuring Dual Oxidation-Based Virus Inactivation

As shown herein under working Example 14, Applicants determined thatmethisazone enhanced inactivation rates while maintaining antigenicityduring dual oxidation-based virus inactivation. To assess the impact ofmethisazone on antigenicity during inactivation, the exemplary modelviruses CHIKV and DENV4 were treated with multiple inactivationapproaches: high concentration H₂O₂ (single oxidation system),dual-oxidation (as described herein), or dual-oxidation withmethisazone. As shown by the ELISA data in FIGS. 19A (Chikungunya virus(CHIKV)) and 19B (dengue virus serotype 4 (DENV4)), the addition ofmethisazone to the dual-oxidation approach maintained or significantlyimproved antigenicity by reducing damage to neutralizing epitopes, whileincreasing the rate of inactivation by approximately 10- to 20-fold.

Chemical Analogs of Methisazone, or Methisazone FunctionalGroups/Substructures or Combinations Thereof, Enhanced Inactivation andMaintenance of Antigenicity During Dual Oxidation-Based ViralInactivation

As shown herein under working Example 15, Applicants determined thatchemical analogs of methisazone, or methisazone functionalgroups/substructures or combinations thereof, enhanced inactivation andmaintenance of antigenicity during dual oxidation-based viralinactivation.

We tested several related compounds to determine if they providedsimilar enhancements to pathogen inactivation for vaccine development(FIGS. 20A-C). As shown with the exemplary model virus DENV4, several ofthese compounds, such as isatin β-thiosemicarbazone and N-propylisatinβ-thiosemicarbazone, demonstrated results similar to methisazoneincluding enhanced rates of inactivation while maintaining superiorantigenicity in the dual-oxidation system. Interestingly, when usingjust the thiosemicarbazide moiety, we still observed enhancement ofinactivation and superior antigenicity, whereas isatin or semicarbazidedo not appear to increase the rate of inactivation, but stilldemonstrate protection of protein antigens from oxidative damage duringinactivation. To explore if the separate major components (functionalgroups/substructures) of methisazone-related compounds could be combinedin order recapitulate optimal inactivation, we tested mixtures ofisatin+thiosemicarbazide or isatin+semicarbazide. Whileisatin+semicarbazide still demonstrated antigen protection, there was noenhancement of virus inactivation. By contrast, isatin+thiosemicarbazideresulted in both rapid inactivation (more rapid than either componentalone) as well as greatly increased antigenicity.

Increasing Levels of Methisazone Relative to the Transition MetalComponent of the Dual Oxidation System Improved the Antigenicity andInactivation Profile of the Dual Oxidation System

As shown herein under working Example 16, Applicants determined thatincreasing levels of methisazone relative to the transition metalcomponent of the dual oxidation system improved the antigenicity andinactivation profile of the dual oxidation system.

The impact of relative concentrations of methisazone and the transitionmetal in the dual-oxidation system (FIG. 21) was examined. We found thatincreasing methisazone concentrations relative to the transition metaldemonstrated concomitant improvements in both retained antigenicity andincreased virus inactivation rates, with a preferred molar ratio of 10:1(methisazone:transition metal).

The Dual Oxidation-Based Inactivation Methods, and Including ThoseFurther Comprising Use of a Methisazone Reagent, Have Broad Utility inthe Development of Advanced Vaccines Against Pathogens Having Either RNAor DNA Genomes, Including but not Lmited to Viral and BacterialPathogens

As discussed above, and shown in the working examples herein, the dualoxidation-based inactivation methods, and including those furthercomprising use of a methisazone reagent, were shown to have utilityacross not only eight viruses in four different viral Families, but alsofor three exemplary bacterial species (e.g., Campylobacter, aGram-negative bacteria, at least a dozen species of which have beenimplicated in human disease, with C. jejuni and C. coli being the mostcommon), Listeria monocytogenes (an exemplary gram-positive bacteria)and Shigella dysenteriae (an exemplary gram-negative bacteria).

According to further aspects, the dual oxidation-based inactivationmethods, and including those further comprising use of a methisazonereagent, have utility for producing highly immunogenic vaccines using,but not limited to the following exemplary microbes:

Viruses. Non-limiting examples of viruses that can be inactivated usingdual oxidation include the following families: Adenoviridae,Alloherpesviridae, Alphaflexiviridae, Alphaherpesvirinae,Alphatetraviridae, Alvernaviridae, Amalgaviridae, Ampullaviridae,Anelloviridae, Arenaviridae, Arteriviridae, Ascoviridae, Asfarviridae,Astroviridae, Autographivirinae, Avsunviroidae, Baculoviridae,Barnaviridae, Benyviridae, Betaflexiviridae, Betaherpesvirinae,Bicaudaviridae, Bidnaviridae, Birnaviridae, Bornaviridae, Bromoviridae,Bunyaviridae, Caliciviridae, Carmotetraviridae, Caulimoviridae,Chordopoxvirinae, Chrysoviridae, Circoviridae, Clavaviridae,Closteroviridae, Comovirinae, Coronaviridae, Coronavirinae,Corticoviridae, Cystoviridae, Densovirinae, Dicistroviridae,Endornaviridae, Entomopoxvirinae, Eucampyvirinae, Filoviridae,Flaviviridae, Fuselloviridae, Gammaflexiviridae, Gammaherpesvirinae,Geminiviridae, Globuloviridae, Gokushovirinae, Guttaviridae,Hepadnaviridae, Hepeviridae, Herpesviridae, Hypoviridae, Hytrosaviridae,Iflaviridae, Inoviridae, Iridoviridae, Leviviridae, Lipothrixviridae,Luteoviridae, Malacoherpesviridae, Marnaviridae, Marseilleviridae,Megabirnaviridae, Mesoniviridae, Metaviridae, Microviridae, Mimiviridae,Myoviridae, Nanoviridae, Narnaviridae, Nimaviridae, Nodaviridae,Nudiviridae, Nyamiviridae, Ophioviridae, Orthomyxoviridae,Orthoretrovirinae, Papillomaviridae, Paramyxoviridae, Paramyxovirinae,Partitiviridae, Parvoviridae, Parvovirinae, Peduovirinae,Permutotetraviridae, Phycodnaviridae, Picobirnaviridae, Picornaviridae,Picovirinae, Plasmaviridae, Pneumovirinae, Podoviridae, Polydnaviridae,Polyomaviridae, Pospiviroidae, Potyviridae, Poxviridae, Pseudoviridae,Quadriviridae, Reoviridae, Retroviridae, Rhabdoviridae, Roniviridae,Rudiviridae, Secoviridae, Sedoreovirinae, Siphoviridae,Sphaerolipoviridae, Spinareovirinae, Spiraviridae, Spounavirinae,Spumaretrovirinae, Tectiviridae, Tevenvirinae, Togaviridae,Tombusviridae, Torovirinae, Totiviridae, Turriviridae, Tymoviridae, andVirgaviridae.

Exemplary viral species include poliovirus, measles virus, mumps virus,parainfluenza virus, Newcastle disease virus, rubella virus, Eastern,Western and Venezuelan Equine Encephalitis Viruses, Lassa virus,lymphocytic choriomeningitis virus, West Nile virus, Dengue virus,Yellow fever virus, Tick-borne encephalitis virus, St. Louisencephalitis virus, Japanese Encephalitis virus, Zika virus, varicellazoster virus, cytomegalovirus, herpes simplex viruses, retrovirusesincluding HIV (human immunodeficiency virus), hepatitis A virus,hepatitis B virus, hepatitis C virus, influenza viruses, rabies virus,molluscum contagiosum, smallpox virus, vaccinia virus, Sindbis virus,swine influenza virus, porcine parvovirus, porcine circovirus,chikungunya virus, porcine reproductive and respiratory syndrome virus,canine distemper virus, canine parvovirus, canine adenovirus Type-2,canine parainfluenzavirus, and canine coronavirus.

Bacteria. Bacterial pathogens can also be inactivated using dualoxidation, and including dual oxidation further comprising use of amethisazone reagent, for use in producing highly immunogenic vaccinecompositions. Non-limiting examples of bacteria that can be inactivatedusing dual oxidation include the following families:Acanthopleuribacteraceae, Acetobacteraceae, Acholeplasmataceae,Acholeplasmataceae, Acidaminococcaceae, Acidilobaceae,Acidimicrobiaceae, Acidimicrobiaceae, Acidithiobacillaceae,Acidobacteriaceae, Acidothermaceae, Actinomycetaceae,Actinopolysporaceae, Actinospicaceae, Actinosynnemataceae,Aerococcaceae, Aeromonadaceae, Akkermansiaceae, Alcaligenaceae,Alcaligenaceae, Alcanivoracaceae, Algiphilaceae, Alicyclobacillaceae,Alteromonadaceae, Anaerolineaceae, Anaeroplasmataceae,Anaeroplasmataceae, Anaplasmataceae, Aquificaceae, Aquificaceae,Archaeoglobaceae, Armatimonadaceae, Aurantimonadaceae, Bacillaceae,Bacteriovoracaceae, Bacteroidaceae, Bacteroidaceae, Bartonellaceae,Bartonellaceae, Bdellovibrionaceae, Beijerinckiaceae, Beijerinckiaceae,Beutenbergiaceae, Bifidobacteriaceae, Blattabacteriaceae,Bogoriellaceae, Brachyspiraceae, Bradyrhizobiaceae, Bradyrhizobiaceae,Brevibacteriaceae, Brevinemataceae, Brucellaceae, Brucellaceae,Burkholderiaceae, Burkholderiaceae, Caldicoprobacteraceae,Caldilineaceae, Caldisericaceae, Caldisphaeraceae, Campylobacteraceae,Cardiobacteriaceae, Carnobacteriaceae, Caryophanaceae, Catalimonadaceae,Catenulisporaceae, Caulobacteraceae, Caulobacteraceae,Celerinatantimonadaceae, Cellulomonadaceae, Chitinophagaceae,Chlamydiaceae, Chlamydiaceae, Chlorobiaceae, Chlorobiaceae,Chloroflexaceae, Christensenellaceae, Chromatiaceae, Chrysiogenaceae,Chrysiogenaceae, Chthonomonadaceae, Clostridiaceae, Cohaesibacteraceae,Colwelliaceae, Comamonadaceae, Comamonadaceae, Conexibacteraceae,Coriobacteriaceae, Coriobacteriaceae, Corynebacteriaceae, Coxiellaceae,Crenotrichaceae, Cryomorphaceae, Cryptosporangiaceae, Cyclobacteriaceae,Cystobacteraceae, Cytophagaceae, Deferribacteraceae, Deferribacteraceae,Defluviitaleaceae, Dehalococcoidaceae, Deinococcaceae, Demequinaceae,Dermabacteraceae, Dermacoccaceae, Dermatophilaceae, Desulfarculaceae,Desulfobacteraceae, Desulfobulbaceae, Desulfohalobiaceae,Desulfomicrobiaceae, Desulfonatronaceae, Desulfovibrionaceae,Desulfurellaceae, Desulfurobacteriaceae, Desulfurococcaceae,Desulfuromonadaceae, Dictyoglomaceae, Dictyoglomaceae, Dietziaceae,Ectothiorhodospiraceae, Ehrlichiaceae, Elusimicrobiaceae,Enterobacteriaceae, Enterococcaceae, Entomoplasmataceae,Entomoplasmataceae, Erysipelotrichaceae, Erysipelotrichaceae,Erythrobacteraceae, Eubacteriaceae, Euzebyaceae, Ferrimonadaceae,Ferroplasmaceae, Fervidicoccaceae, Fibrobacteraceae, Fimbriimonadaceae,Flammeovirgaceae, Flavobacteriaceae, Flexibacteraceae, Francisellaceae,Frankiaceae, Fusobacteriaceae, Fusobacteriaceae, Gaiellaceae,Gallionellaceae, Gemmatimonadaceae, Geobacteraceae, Geodermatophilaceae,Glycomycetaceae, Gordoniaceae, Gracilibacteraceae, Granulosicoccaceae,Hahellaceae, Halanaerobiaceae, Halobacteriaceae, Halobacteroidaceae,Halomonadaceae, Haloplasmataceae, Halothiobacillaceae,Helicobacteraceae, Heliobacteriaceae, Herpetosiphonaceae, Holophagaceae,Holosporaceae, Holosporaceae, Hydrogenophilaceae, Hydrogenophilales,Hydrogenothermaceae, Hydrogenothermaceae, Hyphomicrobiaceae,Hyphomicrobiaceae, Hyphomonadaceae, Iamiaceae, Idiomarinaceae,Ignavibacteriaceae, Intrasporangiaceae, Jiangellaceae, Jonesiaceae,Kiloniellaceae, Kineosporiaceae, Kofleriaceae, Kordiimonadaceae,Ktedonobacteraceae, Lachnospiraceae, Lactobacillaceae, Legionellaceae,Lentisphaeraceae, Leptospiraceae, Leptospiraceae, Leptotrichiaceae,Leuconostocaceae, Listeriaceae, Litoricolaceae, Magnetococcaceae,Marinilabiliaceae, Methanobacteriaceae, Methanocaldococcaceae,Methanocellaceae, Methanococcaceae, Methanocorpusculaceae,Methanomicrobiaceae, Methanopyraceae, Methanoregulaceae,Methanosaetaceae (illegitimate), Methanosarcinaceae,Methanospirillaceae, Methanothermaceae, Methermicoccaceae,Methylobacteriaceae, Methylobacteriaceae, Methylococcaceae,Methylocystaceae, Methylocystaceae, Methylophilaceae, Methylophilaceae,Microbacteriaceae, Micrococcaceae, Micromonosporaceae, Microsphaeraceae,Mooreiaceae, Moraxellaceae, Moritellaceae, Mycobacteriaceae,Mycoplasmataceae, Mycoplasmataceae, Myroidaceae, Myxococcaceae,Nakamurellaceae, Nannocystaceae, Natranaerobiaceae, Nautiliaceae,Neisseriaceae, Nevskiaceae, Nitriliruptoraceae, Nitrosomonadaceae,Nitrospinaceae, Nocardiaceae, Nocardioidaceae, Nocardioidaceae,Nocardiopsaceae, Oceanospirillaceae, Oleiphilaceae, Oligosphaeraceae,Opitutaceae, Orbaceae, Oscillochloridaceae, Oscillospiraceae,Oxalobacteraceae, Oxalobacteraceae, Paenibacillaceae, Parachlamydiaceae,Parachlamydiaceae, Parvularculaceae, Pasteurellaceae, Pasteuriaceae,Patulibacteraceae, Peptococcaceae, Peptostreptococcaceae,Peredibacteraceae, Phaselicystidaceae, Phycisphaeraceae,Phyllobacteriaceae, Phyllobacteriaceae, Picrophilaceae,Piscirickettsiaceae, Planctomycetacea, Planctomycetaceae,Planococcaceae, Polyangiaceae, Porphyromonadaceae, Porphyromonadaceae,Prevotellaceae, Prevotellaceae, Promicromonosporaceae,Propionibacteriaceae, Pseudoalteromonadaceae, Pseudomonadaceae,Pseudonocardiaceae, Psychromonadaceae, Puniceicoccaceae, Pyrodictiaceae,Rarobacteraceae, Rhabdochlamydiaceae, Rhizobiaceae, Rhizobiaceae,Rhodobacteraceae, Rhodobacteraceae, Rhodobiaceae, Rhodobiaceae,Rhodocyclaceae, Rhodospirillaceae, Rhodospirillaceae, Rhodothermaceae,Rickettsiaceae, Rickettsiaceae, Rikenellaceae, Rikenellaceae,Roseiflexaceae, Ruaniaceae, Rubritaleaceae, Rubrobacteraceae,Rubrobacteraceae, Ruminococcaceae, Sandaracinaceae, Sanguibacteraceae,Saprospiraceae, Schleiferiaceae, Segniliparaceae, Serpulinaceae,Shewanellaceae, Simkaniaceae, Simkaniaceae, Sinobacteraceae,Sneathiellaceae, Solimonadaceae, Solirubrobacteraceae,Sphaerobacteraceae, Sphaerobacteraceae, Sphingobacteriaceae,Sphingomonadaceae, Sphingomonadaceae, Spirillaceae, Spirochaetaceae,Spirochetaceae, Spiroplasmataceae, Spiroplasmataceae, Sporichthyaceae,Sporolactobacillaceae, Staphylococcaceae, Streptococcaceae,Streptomycetaceae, Streptosporangiaceae, Succinivibrionaceae,Sulfolobaceae, Sutterellaceae, Synergistaceae, Syntrophaceae,Syntrophobacteraceae, Syntrophomonadaceae, Syntrophorhabdaceae,Thermaceae, Thermithiobacillaceae, Thermoactinomycetaceae,Thermoanaerobacteraceae, Thermoanaerobacteriaceae, Thermococcaceae,Thermodesulfobacteriaceae, Thermodesulfobacteriaceae,Thermodesulfobiaceae, Thermofilaceae, Thermogemmatisporaceae,Thermoleophilaceae, Thermolithobacteraceae, Thermomicrobiaceae,Thermomonosporaceae, Thermoplasmataceae, Thermoproteaceae,Thermosporotrichaceae, Thermotogaceae, Thioalkalispiraceae,Thiotrichaceae, Trueperaceae, Tsukamurellaceae, Turicibacteraceae,Veillonellaceae, Verrucomicrobiaceae, Verrucomicrobiaceae, Vibrionaceae,Victivallaceae, Waddliaceae, Waddliaceae, Williamsiaceae,Xanthobacteraceae, Xanthomonadaceae, Yaniellaceae, Aurantimonadaceae,Cenarchaeaceae,Haliangiaceae, Hydrogenimonaceae, Kordiimonadaceae,Mariprofundaceae, Nitrospiraceae, Parvularculaceae, Procabacteriaceae,Saccharospirillaceae, and Salinisphaeraceae.

Exemplary bacterial species include Campylobacter species (spp.),Shigella spp., Mycobacterium spp., Neisseria spp., Brucella spp.,Borrelia spp., Chlamydia spp., Listeria monocytogenes, Bordatellapertussis, Clostridium spp., Enterococcus spp., Escherichia spp.,Francisella tularensis, Haemophilus influenzae, Helicobacter pylori,Legionella pneumophila, Leptospira interrogans, Streptococcuspneumoniae, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonellaspp., Staphylococcus aureus, and Bacillum anthracis. Gram-positive andGram-negative bacteria, for example, are generally encompassed.

Fungi. Highly immunogenic vaccine compositions can also be produced fromfungal pathogens inactivated using dual oxidation. Exemplary fungalpathogens include: Aspergillus spp., Candida spp, Blastomyces spp.,Coccidioides spp., Cryptococcus spp., Fusarium spp., Histoplasma spp.,Mucorales spp., Pneumocystis spp., Trichophyton spp., Epidermophytonspp., Microsporum spp, Sporothrix spp., Exserohilum spp., andCladosporium spp.

Parasites. The methods disclosed herein can also be used to inactivateparasites (e.g., intracellular parasites) for highly immunogenicvaccines, and especially protozoan parasites, such as Plasmodiumfalciparum and other Plasmodium spp., Leishmania spp., Cryptosporidiumparvum, Entamoeba histolytica, and Giardia lamblia, Trypanosoma spp., aswell as Toxoplasma, Eimeria, Theileria, and Babesia species.

Immunogenic Compositions

Using the disclosed methods, immunogenic compositions, such as vaccinescontaining an inactivated pathogen as also provided. For example, thecomposition (or medicament) can be a lyophilized immunogenic composition(for example, vaccine preparation) containing a pathogen that retainsone or more predominant antigenic epitopes of the biologically activepathogen from which it was prepared. The lyophilized composition may beprepared preservative-free and devoid of any inactivating agent (e.g.,devoid of H₂O₂, etc.). The composition can also be a liquid prepared byreconstituting a lyophilized composition in a pharmaceuticallyacceptable diluent. Optionally, the composition can include a suitableadjuvant that increases the antigenic efficacy of the antigen.

Inactivation with the presently disclosed dual oxidation approach, andincluding those further comprising use of a methisazone reagent, notonly provides improved methods for vaccine production, including forpathogens for which effective vaccines cannot be produced by othermethods (including by peroxide alone), but also provides severaladditional significant benefits as compared to UV inactivation, heatinactivation or inactivation with formaldehyde or betapropiolactone.

First, dual oxidation with hydrogen peroxide plus transition metals ions(Fenton type reaction), and including dual oxidation further comprisinguse of a methisazone reagent, is significantly better than any of theother methods at maintaining immunogenic epitopes. Thus, dual oxidationinactivation, and including dual oxidation further comprising use of amethisazone reagent, produces highly effective immunogenic compositions,such as vaccines, which can be used to produce an immune response thatis far more likely to be protective against subsequent infection by thelive pathogen than are vaccines produced using methods that denature ordestroy immunologically important epitopes.

Second, unlike other chemical inactivating agents, such as formaldehydeor betapropiolactone, the Cu and Fe ions used in the presently discloseddual oxidation methods are not only naturally occurring in subjects, butare present in the reactions in non-toxic amounts. Moreover, residualtransition metals, and/or methisazone reagents, can be removed bydownstream purification using, for example, anion exchangechromatography, flow filtration (e.g., tangential flow filtration), sizeexclusion chromatography, desalting columns, diafiltration, dialysis,ultracentrifugation, sucrose gradient purification, high pressure liquidchromatography (HPLC), etc.

Likewise, any residual hydrogen peroxide can be substantially orcompletely removed from the vaccine composition by either usingsubsequent purification steps as described above for optional transitionmetal removal, or by using lyophilization. For example, a solutioncontaining a pathogen and hydrogen peroxide and transition metal ionscan be dispensed into sterile vials and lyophilized. During thelyophilization process, hydrogen peroxide is removed in vapor form,leaving behind a stable and sterile vaccine composition, which caneasily be stored until it is needed. Lyophilization removes some, mostor even all detectable hydrogen peroxide from the vaccine composition,and where desired produces a vaccine composition that is substantiallyfree of hydrogen peroxide. Lyophilization can be performed byessentially any methods known in the art so long as the temperature ismaintained below that at which heat denaturation of immunogenic epitopesoccurs. Thus, the lyophilization can be performed following pre-freezingof the hydrogen peroxide/pathogen solution) or without pre-freezing (forexample, at ambient temperatures above freezing, e.g., using aSPEED-VAC® concentrator under conditions that maintain the ambienttemperature between about 0-4° C. and about 42° C.). For the purpose ofmanufacturing immunogenic compositions, such as vaccines, foradministration to human or animal subjects, lyophilization is typicallycarried out according to current good manufacturing procedures (cGMP)for the production of vaccines. The inactivation and lyophilization canbe accomplished without any intervening processing step, such asdilution, dialization, centrifugation, or purification. So long as thepathogen/hydrogen peroxide solution is dispensed (or aliquoted) intoclean, sterile containers (e.g., vial, ampules, tubes, etc.) prior tolyophilization, the resulting vaccine composition is sterile, and noadditional preservative need be added prior to administration. Forexample, if the vaccine composition is to be administered in a singledose, the lyophilized vaccine composition is simply suspended (ordissolved) in a pharmaceutically acceptable diluent to produce apreservative-free liquid vaccine composition. In the event that thelyophilized vaccine composition is intended for multiple administrations(for example, multiple sequential administration to a single subject, orone or more administrations to multiple subjects) the diluent caninclude a pharmaceutically acceptable preservative.

If desired, transition metal ions and/or hydrogen peroxide can beremoved by purification steps as described above. For example, residualH₂O₂ and transition metals (e.g., either Cu or Fe) can be removed by useof one or more purification approaches such as tangential flowfiltration, dialysis, desalting columns, ion-exchange chromatography(under conditions that bind the virus but not the residual inactivationcomponents), affinity chromatography, size exclusion chromatography,etc.

Alternatively, sodium bisulfite (NaHSO₃) and/or sodium metabisulfite(Na₂S₂O₅) can both be used to neutralize H₂O₂ (1 mol of metabisulfitebreaks down to two mols of bisulfite, which then reacts directly withH₂O₂).

Na₂S₂O₅+2H₂O→2NaHSO₃+H₂O   (1)

2NaHSO₃+2H₂O₂→2NaHSO₄+2H₂O   (2)

Prior to use, the vaccine can be reconstituted using a pharmaceuticallyacceptable diluent to facilitate delivery by conventional administrationmeans. This enables the production of a sterile vaccine composition thatdoes not contain harmful amounts of toxic and carcinogenic compounds,thereby increasing the safety of the vaccine.

Additionally, following dual oxidation inactivation, or dual oxidationfurther comprising use of a methisazone reagent, there is no need to adda preservative (such as thimerosal) to the resulting vaccinecomposition. The sterile composition can be maintained for long periodsof time (e.g., in the lyophilized state), making addition of potentiallytoxic preservatives unnecessary. Thus, the compositions can be made tobe substantially or completely free of preservatives. Optionally,preservatives can be provided in the composition.

The dual oxidation methods, and including those further comprising useof a methisazone reagent, provide immunogenic compositions, such as avaccine, and thus provide methods for preparing a medicament thatincludes an inactivated pathogen. The methods provide compositions thatcontain an immunologically active noninfectious pathogen that retainspredominant immunological epitopes of the infectious pathogen from whichit is produced. Typically, the inactivated pathogen retains one, or morethan one, immunologically dominant epitopes that elicit a protectiveimmune response against the pathogen. This method is suitable forproducing an immunogenic composition (for example, a vaccine) containinginactivated pathogens, including viruses, bacteria, fungi and parasites,such as intracellular parasites (for example, protozoan parasites).Optionally, the compositions contain more than one species or strain ofpathogen, for example, combination vaccines can be produced using themethods. The compositions can include a plurality of viruses, e.g.,mumps virus, measles virus and rubella virus, and/or other viruses asdisclosed herein. Similarly, the composition can include a plurality ofbacteria, e.g., Campylobacterspecies (spp.), Corynebacteriumdiphtherias, Bordetella pertussis and Clostridium tetani, the causativeagents of diarrhea, diphtheria, whooping cough and tetanus,respectively, and/or other bacterial as disclosed herein. Thecomposition can also include a plurality of pathogens selected fromdifferent classifications (families) of organisms.

The dual oxidation methods involve contacting the pathogen with asolution containing an effective amount of the dual oxidizing agent(e.g., Fenton reagents; hydrogen peroxide (H₂O₂) plus transition metalions), or with the dual oxidizing agent and a methisazone reagent, for aperiod sufficient to render the pathogen noninfectious. Optionally, thepathogen is purified or isolated prior to contacting with the dualoxidizing agent.

Typically, the solution includes at least about 0.001% or 0.002%hydrogen peroxide (wt/vol), and may contain up to about 0.10% hydrogenperoxide. Typically, the solution includes at least 1 μM or 2 μMtransition metal (e.g., CuCl₂). Most typically, at least 0.001% or0.002% hydrogen peroxide (wt/vol) is used in combination with at least 1μM or 2 μM transition metal (e.g., CuCl₂). For example, the solution caninclude about 0.002% hydrogen peroxide (wt/vol), and about 1 μM or 2 μMCuCl₂. In further embodiments, the hydrogen peroxide concentration canbe as low as 0.0001%, or as high 1.0%, in combination withabove-described levels of transition metal. The concentration range oftransition metals can be as low as 0.001 μM, or as high as 1000 μM,again with any of the disclosed levels of hydrogen peroxide. Inreactions comprising a methisazone reagent, the preferred amount ofmethisazone reagent, methisazone analogs, or chemicals representingmethisazone functional groups or methisazone functional substructurescan be as low as 0.01 μM, or as high as 10,000 μM with any of thedisclosed levels of hydrogen peroxide or transition metals.

While the mechanism of the dual-oxidation inactivation was found to besurprisingly different (i.e., found to be proteinconcentration-dependent) than that of simple hydrogen peroxide mediatedoxidation, present Applicants have nonetheless found that the absoluteand/or relative amounts of hydrogen peroxide (wt/vol) and transitionmetal ions (e.g., CuCl₂) can be varied and adjusted to optimizeinactivation while retaining immunogenicity for a broad array ofpathogens. Applicants have found that having two variables (hydrogenperoxide concentration; and transition metal concentration) to vary, andeven three variables in reaction using a methisazone reagent, providesan enhanced fine tuning ability over prior art methods using a singleagent. Moreover, Applicants have surprisingly found (as shown hereinunder the working examples), that the two Fenton components (hydrogenperoxide concentration; and transition metal concentration), as well asthe methisazone reagents in reactions including them, act in synergy toprovide results not achievable using single agents alone. Additionally,combinations of transition metals (e.g., CuCl₂ (Cu²⁺), FeCl₃ (Fe³⁺) orCsCl (Cs)), and methisazone reagents can be employed to exploitsynergistic effects. For example, at CuCl₂ concentrations of 0.05 μM and0.10 μM, increasing FeCl₃ concentrations enhanced antigenicity,indicating synergy with these two metals. These fine-tuning andsynergistic aspects support a broad utility for the presently discloseddual oxidation approach.

The length of time sufficient to completely inactivate a pathogen canvary between several minutes and several hours. For example, thepathogen can be contacted with the dual oxidation solution, or the dualoxidation solution further comprising a methisazone reagent, for a timewithin a range of about 1 hour to 24 hours, or shorter periods.Typically, for dual oxidation reactions, about 20 hours (plus or minus 2hours) is used when using at least 0.001% or 0.002% hydrogen peroxide(wt/vol) is used in combination with at least 1 μM or 2 μM transitionmetal (e.g., CuCl₂). Generally, the length of time sufficient toinactivate the pathogen is dependent on the particular pathogen, and theconcentration of reagents, and one of ordinary skill in the art will beable to empirically determine the concentration of reagents, the lengthof reaction time required, and the reaction temperature, based on thepresent disclosed teachings. In further embodiments, the hydrogenperoxide concentration can be as low as 0.0001%, or as high as 1.0%, incombination with above-described levels of transition metal. Theconcentration range of transition metals can be as low as 0.001 μM, oras high as 1000 μM, again with any of the disclosed levels of hydrogenperoxide. The preferred concentration of the methisazone reagent,methisazone analogs, or chemicals representing methisazone functionalgroups or methisazone functional substructures can be as low as 0.01 μM,or as high as 10,000 μM, with any of the disclosed levels of hydrogenperoxide or transition metals.

The pathogen inactivation can be carried out at any temperature betweenfreezing and the temperature at which immunologically relevant epitopesare denatured. Most commonly, the inactivation process is carried out ator above 4° C. and below about 42° C. For example, it is oftenconvenient to perform the inactivation at room temperature or about 25°C.

Generally speaking, the dual oxidation conditions, including thosefurther comprising a methisazone reagent, are determined to provide ahigh safety margin during the manufacturing process (e.g., up to 100million-fold theoretical excess inactivation) while still maintainingoverall antigenic structure.

The inactivated pathogen can then be stored for prolonged periods (forexample, for more than several months or more than 1 year). The solutioncontaining the inactivated pathogen can then be administered directly toa subject for the purpose of eliciting an immune response against thepathogen, for example, as a vaccine. More commonly, the solutionincluding the inactivated pathogen is further processed or lyophilized,as described above, to produce an immunogenic composition.

The disclosure, therefore, provides immunogenic (e.g., vaccine)compositions produced according to the methods disclosed herein. Forexample, the composition (e.g., a medicament) is a lyophilized and/orpurified composition including an inactivated pathogen that retains oneor more predominant antigenic epitope of the biologically activepathogen. Typically, the composition is substantially or completely freeof any preservative or inactivating agent, such as hydrogen peroxide,formaldehyde or betapropiolactone. In another embodiment, thecomposition is a liquid produced by suspending or dissolving(solubilizing) the lyophilized, or purified composition in apharmaceutically acceptable diluent. Optionally, the diluent contains apreservative. Optionally, the vaccine composition includes an adjuvant.In lyophilized form, the adjuvant can be, for example, an aluminum(e.g., alum or an aluminum salt) adjuvant. Upon preparation of a liquidformulation from the lyophilized vaccine composition, the adjuvant canbe a lipid formulation (e.g., an oil capable of forming an emulsion).The inactivated pathogen genome may comprise RNA or DNA.

Methods for Eliciting an Immune Response in a Subject by Administeringthe Compositions Containing Inactivated Pathogen are also Provided

According to additional aspects, methods of eliciting an immune responseagainst a pathogen by administering the immunogenic compositions areprovided. Typically, the immune response is a protective immune responsethat prevents or reduces infection by one or more pathogens. Forexample, an immune response can be elicited in a subject by preparing acomposition by contacting a pathogen with a solution containing the dualoxidation reagent(s) for a period sufficient to render the pathogennoninfectious (while retaining immunogenicity); and administering thecomposition to a subject, thereby eliciting in the subject an immuneresponse (e.g., a protective immune response) against the pathogen. Insome applications the solution is administered to a subject withoutremoving dual oxidation agent(s) from the solution. In otherapplications, the composition is lyophilized and/or otherwise purifiedas described herein, removing some or all (or substantially all) of thedual oxidation reagent(s). The processed composition can be administeredin powder form (for example, as a dispersed powder or as a pellet, e.g.,using the POWDERJECT® transdermal powder injection device).Alternatively, the lyophilized composition is reconstituted in apharmaceutically acceptable diluent for administration using any methodsuitable for delivering a vaccine to a subject, e.g., intramuscular,intradermal, transdermal, subcutaneous or intravenous injection, oraldelivery, or intranasal or other mucosal delivery of the immunogeniccomposition (e.g., vaccine).

Terms

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9);Kendrew, et al. (eds.), The Encyclopedia of Molecular Biology, publishedby Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratis, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

“An immunogenic composition” or “vaccine composition” or “vaccine” is acomposition of matter suitable for administration to a human or animalsubject that is capable of eliciting a specific immune response, e.g.,against a pathogen. As such, an immunogenic composition or vaccineincludes one or more antigens or antigenic epitopes. The antigen can bein the context of an isolated protein or peptide fragment of a protein,or can be a partially purified preparation derived from a pathogen.Alternatively, the antigen can be in the context of a whole live orinactivated pathogen. Typically, when an immunogenic composition orvaccine includes a live pathogen, the pathogen is attenuated, that is,incapable of causing disease in an immunologically competent subject. Inother cases, an immunogenic composition or vaccine includes a wholeinactivated (or killed) pathogen. The inactivated pathogen can be eithera wild-type pathogenic organism that would otherwise (if notinactivated) cause disease in at least a portion of immunologicallycompetent subjects, or an attenuated or mutant strain or isolate of thepathogen. In the context of this disclosure, the immunogenic and/orvaccine compositions contain a whole (wild-type, attenuated or mutant)pathogen.

An “immune response” is a response of a cell of the immune system, suchas a B cell, T cell, or monocyte, to a stimulus. In some cases, animmune response is a T cell response, such as a CD4+ response or a CD8+response. Alternatively, the response is a B cell response, and resultsin the production of specific antibodies. In some cases, the response isspecific for a particular antigen (that is, an “antigen-specificresponse”). If the antigen is derived from a pathogen, theantigen-specific response is a “pathogen-specific response.” A“protective immune response” is an immune response that inhibits adetrimental function or activity of a pathogen, reduces infection by apathogen, or decreases symptoms (including death) that result frominfection by the pathogen. A protective immune response can be measured,for example, by the inhibition of viral replication or plaque formationin a plaque reduction assay or ELISA-neutralization assay, or bymeasuring resistance to viral challenge in vivo.

An “immunologically effective amount” is a quantity of a compositionused to elicit an immune response in a subject. In the context of avaccine administration, the desired result is typically a protectivepathogen-specific immune response. However, to obtain protectiveimmunity against a pathogen in an immunocompetent subject, multipleadministrations of the vaccine composition are commonly required. Thus,in the context of this disclosure, the term immunologically effectiveamount encompasses a fractional dose that contributes in combinationwith previous or subsequent administrations to attaining a protectiveimmune response.

An “antigen” is a compound, composition, or substance that can stimulatethe production of antibodies and/or a T cell response in an animal,including compositions that are injected, absorbed or otherwiseintroduced into an animal. The term “antigen” includes all relatedantigenic epitopes. The term “epitope” or “antigenic determinant” refersto a site on an antigen to which B and/or T cells respond.

The “predominant antigenic epitopes” are those epitopes to which afunctionally significant host immune response, e.g., an antibodyresponse or a T-cell response, is made. Thus, with respect to aprotective immune response against a pathogen, the predominant antigenicepitopes are those antigenic moieties that when recognized by the hostimmune system result in protection from disease caused by the pathogen.

The term “antigenicity” refers to the relative maintenance ofimmunogenic epitope structure(s) as determined, for example, by variousin vitro measurements, such as binding of specific monoclonal antibodiesor hemagglutination assays. “Antigenicity” in the in vivo context istypically referred to herein as “immunogenicity”. An “adjuvant” is anagent that enhances the production of an immune response in anon-specific manner. Common adjuvants include suspensions of minerals(e.g., alum, aluminum hydroxide, aluminum phosphate) onto which antigenis adsorbed; or water-in-oil emulsions in which an antigen solution isemulsified in oil (MF-59, Freund's incomplete adjuvant). Additionaldetails regarding various adjuvants can be found in Derek O'HaganVaccine Adjuvants: Preparation Methods and Research Protocols (Methodsin Molecular Medicine) Humana Press, 2000.

The term “pathogen” as used herein refers to an organism having eitheran RNA or DNA genome, and encompasses viruses (both RNA and DNAgenome-based), bacteria (DNA genome-based, both Gram-positive andGram-negative), fungi, and parasites. In particular preferred aspects,“pathogen” refers to an organism having either an RNA or DNA genome, andencompasses viruses (both RNA and DNA genome-based), and bacteria(DNA-genome based, both Gram-positive and Gram-negative).

The term “whole pathogen” refers to a pathogenic organism, such as avirus, a bacterium, a fungus or a parasite, that includes all orsubstantially all of the constituents of the infectious form of theorganism. Typically, a whole pathogen is capable of replication. Theterm “whole pathogen” is nonetheless distinct from the term “wild-type”pathogen, and the term “whole pathogen” encompasses wild-type as well asattenuated and other mutant forms of the pathogenic organism. Thus, awhole pathogen can be an attenuated pathogen incapable of causingdisease in an immunocompetent host, but nonetheless including all orsubstantially all of the constituents of an infectious pathogen.Similarly, a whole pathogen can be a mutant form of the pathogen,lacking one or more intact (wild-type) genes, and/or proteins. Thepathogen genome may comprise RNA or DNA.

An “inactivated pathogen” is a whole pathogen that has been renderedincapable of causing disease (e.g., rendered noninfectious) byartificial means. Typically, an inactivated pathogen is a “killedpathogen” that is incapable of replication. A pathogen is noninfectiouswhen it is incapable of replicating or incapable of replicating tosufficient levels to cause disease.

An “immunogenically active vaccine”, as used herein in connection withApplicants' methods, is a pathogen inactivated by the disclosed methodsthat is capable of eliciting an immune response when introduced into animmunologically competent subject. The immune response produced inresponse to exposure to an immunogenically active vaccine comprising theinactivated pathogen as disclosed herein is preferably identical,substantially identical, or superior with respect to that produced bythe predominant antigenic epitopes of the respective infectiouspathogen.

“Hydrogen peroxide” (H₂O₂) is an exemplary preferred oxidizing agentwith a standard electrode potential of 1.78 volts. For the purpose ofconsistency, the proportion of hydrogen peroxide in a solution, as inthe working Examples disclosed herein, is given as weight per volume(wt/vol). For example 0.01% H₂O₂ refers to H₂O₂ being present at 0.01%wt/vol.

A “dual oxidizing agent” as used herein refers to a Fenton-type dualoxidation reagent comprising hydrogen peroxide and at least onetransition metal (e.g., CuCl₂ (Cu²⁺), FeCl₃ (Fe³) or CsCl (Cs⁺)).

A “solution comprising the dual oxidizing agent(s)” includes thecombination of any mixture of a solvent and dual oxidizing agent(s).Most commonly, in the context of the methods disclosed herein thesolvent is water, e.g., deionized water, or an aqueous buffered saltsolution. Typically, the term solution includes liquid phase solutions.For the purpose of consistency, the proportion of hydrogen peroxide in asolution is given as weight per volume (wt/vol).

The phrase “substantially free of hydrogen peroxide” indicates that nomore than trace amounts (amounts empirically detectable as background)are present in the composition.

The verb “lyophilize” means to freeze-dry under vacuum. The process istermed “lyophilization.” In some cases, the sample to be dried (e.g.,dehydrated) is frozen prior to drying. In other cases, the material tobe dried is subjected to the drying process without prior phase change.During the process of lyophilization, evaporation of the solvent resultsin cooling of the sample to temperatures below the melting temperatureof the solvent/solute mixture resulting in freezing of the sample.Solvent is removed from the frozen sample by sublimation. A product thathas undergone lyophilization is “lyophilized.” As used in thisdisclosure the term lyophilization also encompasses functionallyequivalent procedures that accelerate the drying process withoutexposing the sample to excessive heat, specifically including: spraydrying and spray freeze-drying.

The term “methisazone” and “methisazone analog” as used herein inparticular aspects refers to compounds having the following formula:

wherein R₁ is independently H or lower alkyl (e.g., C1-C4 alkyl)optionally substituted with —OH, for example, wherein R₁ is H, —CH₃, orpropyl, etc.; wherein R₂ is independently H, lower alkyl (e.g., C1-C2alkyl) optionally substituted with —OH, or aryl; wherein X isindependently H or halogen (e.g., I, Br, Cl, F); and salts, includingpharmaceutically acceptable salts, thereof. Preferably, wherein X and R₂are H; and wherein R₁ is H (isatin β-thiosemicarbazone), —CH₃(N-methyl-isatin β-thiosemicarbazone (methisazone)), or propyl(N-propyl-isatin β-thiosemicarbazone). Preferably, methisazone is used:

The term “methisazone functional group” or “methisazone functionalsubstructure” as used herein in particular aspects refers to compoundshaving the following formulae:

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH, for example, wherein X is H and wherein R₁ is H(isatin) or —CH₃ (N-methyl-isatin), or propyl (N-propyl-isatin), etc.;wherein X is independently H or halogen (e.g., I, Br, Cl, F); and salts,including pharmaceutically acceptable salts, thereof;

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH, for example, wherein X is H and wherein R₁ is H(indole, 2,3-dione, 3-hydrazone) etc.; wherein X is independently H orhalogen (e.g., I, Br, Cl, F); wherein R₂ is independently H, lower alkyl(e.g., C1-C2 alkyl) optionally substituted with —OH, or aryl; and salts,including pharmaceutically acceptable salts, thereof and

wherein R₂ and R₃ are independently H, lower alkyl (e.g., C1-C2 alkyl)optionally substituted with —OH, or aryl; and salts, includingpharmaceutically acceptable salts, thereof and combinations thereof.

In particular aspects, the following combinations of “methisazonefunctional group” or “methisazone functional substructure” are used:

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl), for example, whereinR₁ is H (isatin) or —CH₃ (N-methyl-isatin), or propyl (N-propyl-isatin),etc., and salts, including pharmaceutically acceptable salts, thereof.

In particular aspects, the following combination of “methisazonefunctional groups” or “methisazone functional substructures” is used:

In the context of this disclosure “room temperature” refers to anytemperature within a range of temperatures between about 16° C.(approximately 61° F.) and about 25° C. (approximately 77° F.).Commonly, room temperature is between about 20° C. and 22° C. (68°F.-72° F.). Generally, the term room temperature is used to indicatethat no additional energy is expended cooling (e.g., refrigerating) orheating the sample or ambient temperature.

A “preservative” is an agent that is added to a composition to preventdecomposition due to chemical change or microbial action. In the contextof vaccine production, a preservative is typically added to preventmicrobial (e.g., bacterial and fungal) growth. The most commonpreservative used in vaccine production is thimerosal, a mercurycontaining organic compound. Thus, the term “preservative-free”indicates that no preservative is added to (or present in) thecomposition.

The term “purification” (e.g., with respect to a pathogen or acomposition containing a pathogen) refers to the process of removingcomponents from a composition, the presence of which is not desired.Purification is a relative term, and does not require that all traces ofthe undesirable component be removed from the composition. In thecontext of vaccine production, purification includes such processes ascentrifugation, dialization, ion-exchange chromatography, andsize-exclusion chromatography, affinity-purification, precipitation andother methods disclosed herein (e.g., lyophilization, etc). Suchpurification processes can be used to separate the inactiavated pathogencomponents from the reagents used to inactivate the respective pathogenas disclosed herein. For example hydrogen peroxide, metal reagents,“methisazone”, “methisazone analogs” “methisazone functional groups” or“methisazone functional substructures” can be separated from theinactiavated pathogen components to provide purified vaccinecompositions. For example, residual methisazone, methisazone analogs, orchemicals representing methisazone functional groups or methisazonefunctional substructures may range from 0.0001 to 10 mM when used forvaccine antigen preparation. A range of standard purification techniquesmay be used to remove or separate these residual components from vaccineantigen prior to final formulation, including, but not limited to,affinity chromatography, ion-exchange chromatography,mixed-mode/multimodal chromatography, gel filtration/size-exclusionchromatography, desalting chromatography, tangential flowfiltration/diafiltration, density-gradient centrifugation, centrifugalfiltration, dialysis, vaccine antigen precipitation or vaccine antigenadsorption.

The adjective “pharmaceutically acceptable” indicates that the subjectis physiologically acceptable for administration to a subject (e.g., ahuman or animal subject). Remington's Pharmaceutical Sciences, by E. W.Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describescompositions and formulations (including diluents) suitable forpharmaceutical delivery of therapeutic and/or prophylactic compositions,including vaccines.

In general, the nature of the diluent will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In certain formulations (for example, solid compositions, suchas powder, pill, tablet, or capsule forms), a liquid diluent is notemployed. In such formulations, non-toxic solid carriers can be used,including for example, pharmaceutical grades of mannitol, lactose,starch or magnesium stearate.

The phrase “Good Manufacturing Practice” or “GMP” with respect tomethods and procedures employed in vaccine production refer specificallyto the set of methods, protocols and procedures established by theUnited States Food and Drug Administration (FDA). Similarrecommendations and guidelines are promulgated by the World HealthOrganization. The abbreviation “cGMP” specifically designates thoseprotocols and procedures that are currently approved by the FDA (e.g.,under 21 Code of Federal Regulations, parts 210 and 211, available onthe world wide web at fda.gov/cder/dmpq). With time cGMP compliantprocedures may change. Any methods disclosed herein can be adapted inaccordance with new cGMP requirements as mandated by the FDA.

Inactivation of Pathogens

To inactivate a pathogen using dual oxidizing agent(s), including thosefurther comprising a methisazone reagent, the live pathogen is grown toa desired density (e.g., saturation density in culture), according toany procedures acceptable in the art for growing (e.g., culturing thespecific organism). Typically, for cellular pathogens, it is desirableto culture the pathogen to stationary phase; as such organisms aregenerally more resistant to stresses in subsequent processing than thoseharvested at logarithmic phase. Growth in culture can be monitored usingmethods known in the art, such as measuring optical density of theculture using spectrophotometry. When the pathogen is a virus, growthcan monitored by titering the virus using standard methods establishedfor the selected virus. For example, methods for growing animal virusescan be found, for example, in DNA Viruses: A Practical Approach, Alan J.Cann (ed.) Oxford University Press, 2000; Robinson and Cranage (eds.)Vaccine Protocols (Methods in Molecular Medicine) Humana Press, 2003,and references cited therein. Methods for culturing pathogenic bacteriaare also known in the art, and can be found in Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, ed. Sambrook, et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Methods forculturing parasites, such as malaria, are also known in the art, e.g.,Denise Doolan (ed.) Malaria Methods and Protocols (Methods in MolecularMedicine) Humana Press, 2002, and references cited therein.

Typically, the pathogenic organisms can have RNA or DNA genomes (e.g.,viruses, bacteria, fungus, or parasites) and are purified from themedium in which they are grown or cultured, and in the case of pathogensthat replicate inside a cell are purified from the other cellularcomponents. For example, the relative concentration of non-pathogencomponents of a suspension including pathogens can be decreased by atleast 50%, such as about 70%, or by as much as 80%, or even by 90%, 95%or more, relative to a crude preparation of pathogen. Intracellularpathogens, such as viruses, can be isolated or purified from the variouscomponents of the cells they infect by various methods known in the art.

For example, viruses for vaccine production are typically grown undercontrolled conditions in a certified cell line using biologically andchemically defined culture medium according to cGMP procedures. Cellsare usually infected with virus at an appropriate multiplicity ofinfection (MOI), and the cells are maintained in culture underconditions and for a period of time sufficient to permit replication ofthe virus to high titer. The cells are then harvested by centrifugation(following release from the culture surface in the case of adherentcells), and resuspended in an appropriately buffered solution. Tofacilitate recovery, the buffered solution is typically hypotonic withrespect to the cells, causing the cells to swell. Optionally, the cellsuspension is agitated periodically to ensure a more uniform exposure ofthe cells to the hypotonic solution. The cells are then lysed, forexample, by homogenization, to release the virus. The lysate iscentrifuged to remove large particulate matter, such as cell nuclei, andthe supernatant is filtered to remove additional cellular debris. Thevirus can then be further purified by layering the filtered supernatantonto a suitable separation medium, such as a sucrose density gradient.Optionally, the nuclear pellet can be further processed to increaseviral yield. The nuclear pellet is resuspended again in hypotonic bufferand homogenized. The nuclear lysate is centrifuged and the resultingsupernatant is filtered prior to layering onto separation medium.Optionally, the two viral suspensions are combined to achieve anapproximately equal volume separation gradient. The separationmedium/virus suspension is then processed by ultracentrifugation (e.g.,at 55,000×g for 1-1.5 hours at 4° C. Virus is collected into a pellet bythis process whereas membranous cellular debris remains at theinterface. The supernatant is removed (typically by aspiration) and thepellet is resuspended in buffer. The purified virus can then beevaluated for recovery and viability (for example by determining proteinconcentration and by plaque assays, respectively). If desired therecovered virus can be frozen and stored until use.

Similar procedures are known in the art for purifying non-viralpathogens, such as intracellular parasites (for example, protozoanparasites, including Plasmodium falciparum and other Plasmodium species,Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, andGiardia lamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesiaspecies).

Reconstitution and Administration

Immunogenic compositions, such as vaccines, that are produced as powders(e.g., lyophilized powders) are typically mixed with a liquid foradministration. This process is known as “reconstitution,” and theliquid used is commonly referred to as a “diluent.” For purposes ofadministration, especially to human subjects, it is important that thediluent be a pharmaceutically acceptable formulation. Reconstitution ofthe lyophilized composition is typically carried out using a sterilesyringe and needle for each vial of diluent. The correct diluent foreach type and batch is used to ensure adequate potency, safety andsterility of the resulting mixture. Diluents are specifically designedto optimize delivery and efficacy of the selected composition. Commondiluents include such additives as: stabilizers to improve heatstability of the vaccine; agents, such as surfactants, to assist indissolving the powder into a liquid; and buffers to ensure the correctacidic balance of the reconstituted composition. Optionally, the diluentcan contain a preservative (e.g., a bactericide and/or a fungicide) tomaintain sterility after reconstitution. Preservatives are typicallyrequired (e.g., by the FDA) when the composition is reconstituted in amulti-dose formulation.

Administration of Immunogenic Compositions Such as Vaccines (TherapeuticMethods)

The immunogenic compositions (such as vaccine or other medicaments)disclosed herein can be administered to a subject to elicit an immuneresponse against a pathogen. Most commonly, the compositions areadministered to elicit a prophylactic immune response against apathogenic organism to which the subject has not yet been exposed. Forexample, vaccine compositions including dual oxidation-inactivatedpathogens can be administered as part of a localized or wide-spreadvaccination effort. An immune response elicited by administration ofsuch vaccine compositions typically includes a neutralizing antibodyresponse, and can in addition include a T cell response, e.g., acytotoxic T cell response that targets cellular pathogens. Accordingly,methods for making a medicament or pharmaceutical composition containingdual oxidation-inactivated pathogens are included herein. Thepharmaceutical compositions (medicaments) include at least one pathogeninactivated by contact with a solution containing the dual oxidizingagent(s), or by contact with the dual oxidizing agents furthercomprising a methisazone reagent, in a pharmaceutically acceptablecarrier or excipient.

In some cases, the immunogenic composition can include a combination ofpathogens, such as a combination of viruses (for example mumps virus,measles virus, rubella virus), or a combination of bacteria (forexample, Campylobacter species (spp.), Corynebacterium diptheriae,Bordatella pertussis, and Clostridium tetani), or a combination ofpathogens selected from different classes of organisms, e.g., one ormore viruses and one or more bacteria, one or more bacteria and one ormore parasites, and the like.

The quantity of pathogen included in the composition is sufficient toelicit an immune response when administered to a subject. For example,when administered to a subject in one or more doses, a vaccinecomposition containing an inactivated pathogen favorably elicits aprotective immune response against the pathogen. A dose of the vaccinecomposition can include at least about 0.1% wt/wt inactivated pathogento about 99% wt/wt inactivated pathogen, with the balance of the vaccinecomposition is made up of pharmaceutically acceptable constituents, suchas a pharmaceutically acceptable carrier and/or pharmaceuticallyacceptable diluent. Guidelines regarding vaccine formulation can befound, e.g., in U.S. Pat. Nos. 6,890,542, and 6,651,655. In onespecific, non-limiting example the vaccine composition (medicament)includes at least about 1%, such as about 5%, about 10%, about 20%,about 30%, or about 50% wt/wt inactivated pathogen. As will be apparentto one of ordinary skill in the art, the quantity of pathogen present inthe vaccine formulation depends on whether the composition is a liquidor a solid. The amount of inactivated pathogen in a solid compositioncan exceed that tolerable in a liquid composition. The amount ofinactivated pathogen can alternatively be calculated with respect to thecomparable amount of a live or inactivated pathogen required to give animmune response. For example, a dosage equivalent in viral particles tofrom about 10⁶ to about 10¹² plaque forming units (PFU) of live orattenuated virus can be included in a dose of the vaccine composition.Similarly, a vaccine composition can include a quantity of inactivatedpathogen (e.g., with RNA or DNA genome), such as virus, bacteria, fungusor parasite equivalent to between about 10³ to about 10¹⁰ liveorganisms. Alternatively, the dosage can be provided in terms of proteincontent or concentration. For example, a dose can include fromapproximately 0.1 μg, such as at least about 0.5 μg protein. Forexample, a dose can include about 1 μg of an isolated or purified virusor other pathogen up to about 100 μg, or more of a selected pathogen.Although the equivalent doses in infectious units (e.g., PFU) can varyfrom pathogen to pathogen, the appropriate protein dose can beextrapolated (for example, from PFU) or determined empirically. Forexample, in a typical preparation, 1 μg of purified vaccinia virus isequivalent to approximately 2×10⁶ PFU. Similar conversions can bedetermined for any pathogen of interest.

Typically, preparation of a vaccine composition (medicament) entailspreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals. Typically, the pharmaceutical composition containsappropriate salts and buffers to render the components of thecomposition stable and allow for appropriate processing and presentationof the vaccine antigen by antigen presenting cells. Such components canbe supplied in lyophilized form, or can be included in a diluent usedfor reconstitution of a lyophilized form into a liquid form suitable foradministration. Alternatively, where the inactivated pathogen isprepared for administration in a solid state (e.g., as a powder orpellet), a suitable solid carrier is included in the formulation.

Aqueous compositions typically include an effective amount of theinactivated pathogen dispersed (for example, dissolved or suspended) ina pharmaceutically acceptable diluent or aqueous medium. The phrase“pharmaceutically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or otherundesirable reaction when administered to a human or animal subject. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, isotonic and absorption delayingagents and the like. Optionally, a pharmaceutically acceptable carrieror diluent can include an antibacterial, antifungal or otherpreservative. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with production of an immuneresponse by an inactivated pathogen, its use in the immunogeniccompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. For example, certainpharmaceutical compositions can include the inactivated pathogen in anaqueous diluent, mixed with a suitable surfactant, such ashydroxypropylcellulose. Dispersions also can be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof and in oils. In somecases (for example, when liquid formulations are deemed desirable, orwhen the lyophilized vaccine composition is reconstituted for multipledoses in a single receptacle), these preparations contain a preservativeto prevent the growth of microorganisms.

Pharmaceutically acceptable carriers, excipients and diluents are knownto those of ordinary skill in the described, e.g., in Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of inactivated pathogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example, sodiumacetate or sorbitan monolaurate.

For example, the pharmaceutical compositions (medicaments) can includeone or more of a stabilizing detergent, a micelle-forming agent, and anoil. Suitable stabilizing detergents, micelle-forming agents, and oilsare detailed in U.S. Pat. No. 5,585,103; U.S. Pat. No. 5,709,860; U.S.Pat. No. 5,270,202; and U.S. Pat. No. 5,695,770. A stabilizing detergentis any detergent that allows the components of the emulsion to remain asa stable emulsion. Such detergents include polysorbate, 80 (TWEEN80)(Sorbitan-mono-9-octadecenoate-poly(oxy-1,2-ethanediyl; manufactured byICI Americas, Wilmington, Del.), TWEEN 40™, TWEEN 20™, TWEEN 60™,Zwittergent™3-12, TEEPOL HB7™, and SPAN 85™. These detergents areusually provided in an amount of approximately 0.05 to 0.5%, such as atabout 0.2%. A micelle forming agent is an agent which is able tostabilize the emulsion formed with the other components such that amicelle-like structure is formed. Such agents generally cause someirritation at the site of injection in order to recruit macrophages toenhance the cellular response. Examples of such agents include polymersurfactants described by, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110,1977, and Hunter et al., J. Immunol 129:1244, 1981, and such agents asPLURONIC™ L62LF, L101, and L64, PEG1000, and TETRONIC™ 1501, 150R1, 701,901, 1301, and 130R1. The chemical structures of such agents are wellknown in the art. In one embodiment, the agent is chosen to have ahydrophile-lipophile balance (HLB) of between 0 and 2, as defined byHunter and Bennett, J. Immun. 133:3167, 1984. The agent can be providedin an effective amount, for example between 0.5 and 10%, or in an amountbetween 1.25 and 5%.

The oil included in the composition is chosen to promote the retentionof the pathogen in oil-in-water emulsion, and preferably has a meltingtemperature of less than 65° C., such that emulsion is formed either atroom temperature, or once the temperature of the emulsion is adjusted toroom temperature. Examples of such oils include squalene, Squalane,EICOSANE™, tetratetracontane, glycerol, and peanut oil or othervegetable oils. In one specific, non-limiting example, the oil isprovided in an amount between 1 and 10%, or between 2.5 and 5%. The oilshould be both biodegradable and biocompatible so that the body canbreak down the oil over time, and so that no adverse effects are evidentupon use of the oil.

Optionally, the pharmaceutical compositions or medicaments can include asuitable adjuvant to increase the immune response against the pathogen.As used herein, an “adjuvant” is any potentiator or enhancer of animmune response. The term “suitable” is meant to include any substancewhich can be used in combination with the selected pathogen to augmentthe immune response, without producing adverse reactions in thevaccinated subject. Effective amounts of a specific adjuvant may bereadily determined so as to optimize the potentiation effect of theadjuvant on the immune response of a vaccinated subject. For example,suitable adjuvants in the context of vaccine formulations include 03%-5% (e.g., 2%) aluminum hydroxide (or aluminum phosphate) and MF-59 oilemulsion (0.5% polysorbate 80 and 0.5% sorbitan trioleate. Squalene(5.0%) aqueous emulsion) is another adjuvant which has been favorablyutilized in the context of vaccines. For example, the adjuvant can be amixture of stabilizing detergents, micelle-forming agent, and oilavailable under the name Provax® (DEC Pharmaceuticals, San Diego,Calif.). An adjuvant can also be an immunostimulatory nucleic acid, suchas a nucleic acid including a CpG motif. Other adjuvants includemineral, vegetable or fish oil with water emulsions, incomplete Freund'sadjuvant, E. coli J5, dextran sulfate, iron sulfate, iron oxide, sodiumalginate, Bacto-Adjuvant, certain synthetic polymers such as Carbopol(BF Goodrich Company, Cleveland, Ohio), poly-amino acids and co-polymersof amino acids, saponin, carrageenan, REGRES SIN (Vetrepharm, Athens,Ga.), AVRIDINE (N, N-dioctadecyl-N′,N′-bis(2-hydroxyethyl)-propanediamine), long chain polydispersed .beta.(1,4) linked mannan polymers interspersed with 0-acetylated groups (e.g.ACEMANNAN), deproteinized highly purified cell wall extracts derivedfrom non-pathogenic strain of Mycobacterium species (e.g., EQUIMUNE,Vetrepharm Research Inc., Athens Ga.), Mannite monooleate, paraffin oiland muramyl dipeptide. A suitable adjuvant can be selected by one ofordinary skill in the art.

The pharmaceutical compositions (medicaments) can be prepared for use intherapeutic or prophylactic regimens (e.g., vaccines) and administeredto human or non-human subjects to elicit an immune response against oneor more pathogens. For example, the compositions described herein can beadministered to a human (or non-human) subject to elicit a protectiveimmune response against one or more pathogens. To elicit an immuneresponse, a therapeutically effective (e.g., immunologically effective)amount of the inactivated pathogen is administered to a subject, such asa human (or non-human) subject.

A “therapeutically effective amount” is a quantity of a composition usedto achieve a desired effect in a subject being treated. For instance,this can be the amount necessary to stimulate an immune response, toprevent infection, to reduce symptoms, or inhibit transmission of apathogen. When administered to a subject, a dosage will generally beused that will achieve target tissue concentrations (for example, inantigen presenting cells) that is empirically determined to achieve anin vitro effect. Such dosages can be determined without undueexperimentation by those of ordinary skill in the art.

An immunogenic composition, such as a vaccine composition containing aninactivated pathogen, can be administered by any means known to one ofskill in the art, such as by intramuscular, subcutaneous, or intravenousinjection, but even oral, nasal, and transdermal mutes are contemplated.In one embodiment, administration is by subcutaneous or intramuscularinjection. To extend the time during which the inactivated pathogen isavailable to stimulate a response, the peptide can be provided as anoily injection, as a particulate system, or as an implant. Theparticulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. A particulate carrierbased on a synthetic polymer has been shown to act as an adjuvant toenhance the immune response, in addition to providing a controlledrelease.

As an alternative to liquid formulations, the composition can beadministered in solid form, e.g., as a powder, pellet or tablet. Forexample, the vaccine composition can be administered as a powder using atransdermal needleless injection device, such as the helium-poweredPOWDERJECT® injection device. This apparatus uses pressurized helium gasto propel a powder formulation of a vaccine composition, e.g.,containing an inactivated pathogen, at high speed so that the vaccineparticles perforated the stratum corneum and land in the epidermis.

Polymers can be also used for controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston, etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema, et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri, etal., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (e.g., U.S. Pat. No.5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat.No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; andU.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat.No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No. 533096).

In specific, non-limiting examples, the inactivated pathogen (e.g., aparasite, such as a protozoan parasite, or a bacterial pathogen) isadministered to elicit a cellular immune response (e.g., a cytotoxic Tlymphocyte (CTL) response). A number of means for inducing cellularresponses, both in vitro and in vivo, are known. Lipids have beenidentified as agents capable of assisting in priming CTL responses invivo against various antigens. For example, as described in U.S. Pat.No. 5,662,907, palmitic acid residues can be attached to the alpha andepsilon amino groups of a lysine residue and then linked (e.g., via oneor more linking residues, such as glycine, glycine-glycine, serine,serine-serine, or the like) to an immunogenic peptide or protein. Thelipidated peptide can then be injected directly in a micellar form,incorporated in a liposome, or emulsified in an adjuvant. As anotherexample, E coli lipoproteins, such astripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime tumorspecific CTL when covalently attached to an appropriate peptide (see,Deres et al., Nature 342:561, 1989). Further, as the induction ofneutralizing antibodies can also be primed with the same moleculeconjugated to a peptide which displays an appropriate epitope, twocompositions can be combined to elicit both humoral and cell-mediatedresponses where that is deemed desirable.

Dosages of inactivated pathogen are administered that are sufficient toelicit an immune response, e.g., a protective immune response, in asubject. With respect to viral pathogens, the dosage is typicallycalculated based on the amount of biological matter equivalent to aspecified titer of infectious (e.g., virulent or attenuated) virus. Forexample, a dose equivalent to about 10⁶, or about 10⁷, or about 10⁸, orabout 10⁹, or about 10¹⁰, or about 10¹¹ or about 10¹², or even more livevirus per dose can be administered to elicit an immune response in asubject. In some cases, the dose includes an amount in excess of theamount of a live virus utilized to elicit an immune response, becausethe inactivated vaccine is incapable of increasing in number afteradministration into the subject. When calculating the amount of acellular pathogen, e.g., a bacteria, a fungus or a parasite, the amountcan be calculated by comparison to a dose of live bacteria, e.g., fromabout 10³ cells or organisms to about 10¹⁰ live organisms, depending onthe formulation. For example, the dose can include at least about 100nanograms (or 200 nanograms, or 500 nanograms, or 1 microgram) ofprotein antigen per dose to about 25 mg (e.g., about 10 mg, or about 15mg, or about 20 mg), or even more of an inactivated pathogen. Typicallythe vaccine composition includes additional pharmaceutically acceptableconstituents or components. Accordingly, the vaccine composition caninclude at least about 0.1% wt/wt inactivated pathogen to about 99%wt/wt inactivated pathogen, with the balance of the vaccine compositionis made up of pharmaceutically acceptable constituents, such as a one ormore pharmaceutically acceptable carrier, pharmaceutically acceptablestabilizer and/or pharmaceutically acceptable diluent. Guidelinesregarding vaccine formulation can be found, e.g., in U.S. Pat. Nos.6,890,542, and 6,651,655. Doses can be calculated based on proteinconcentration (or infectious units, such as PRJ, of infectious unitequivalents). The optimal dosage can be determined empirically, forexample, in preclinical studies in mice and non-human primates, followedby testing in humans in a Phase I clinical trial. Actual methods forpreparing administrable compositions will be known or apparent to thoseskilled in the art and are described in more detail in such publicationsas Remington's Pharmaceutical Sciences, 19th Ed., Mack PublishingCompany, Easton, Pa., 1995.

Typically, but not always, the vaccine compositions are administeredprior to exposure of a subject to a pathogen, e.g., as a vaccine.Vaccine compositions can be prepared by inactivating a wide range ofpathogens using dual oxidizing conditions, or using dual oxidizingconditions further comprising a methisazone reagent(s), according to themethods described herein. For example, vaccine compositions can beprepared by inactivating a pathogenic virus with a solution containingdual oxidizing reagent(s), or with a solution containing dual oxidizingreagent(s) further comprising a methisazone reagent(s). Non-limitingexamples of viruses that can be inactivated by the dual oxidationmethods for vaccine production are disclosed herein.

Bacterial pathogens can also be inactivated using dual oxidizingreagent(s), or using dual oxidizing conditions further comprising amethisazone reagent(s), for use in vaccine compositions. Non-limitingexamples of bacteria that can be inactivated by the dual oxidationmethods for vaccine production are disclosed herein.

Vaccine compositions can also be produced from fungal pathogensinactivated using dual oxidizing reagent(s), or using dual oxidizingconditions further comprising a methisazone reagent(s). Non-limitingexamples of fungal pathogens that can be inactivated by the dualoxidation methods for vaccine production are disclosed herein.

Vaccine compositions can also be produced from parasitic pathogensinactivated using dual oxidizing reagent(s), or using dual oxidizingconditions further comprising a methisazone reagent(s). Non-limitingexamples of parasitic pathogens that can be inactivated by the dualoxidation methods for vaccine production are disclosed herein.

It will be apparent that the precise details of the methods orcompositions described can be varied or modified without departing fromthe spirit of the described invention. The following examples areprovided to illustrate certain particular features and/or embodiments.These examples should not be construed to limit the invention to theparticular features or embodiments described. Each of the referencescited below is incorporated by reference for all purposes.

EXAMPLE 1 Standard H₂O ₂-Based Inactivation was Shown to InactivateCHIKV, but Also Damaged CHIKV-Specific Neutralizing Epitopes and Failedto Induce Neutralizing Responses In Vivo Following Vaccination

FIG. 2 shows that standard H₂O₂-based inactivation disruptsCHIKV-specific neutralizing epitopes and fails to induce neutralizingresponses in vivo following vaccination.

In FIG. 2A, Chikungunya virus (CHIKV) samples received no treatment(Live CHIKV) or were treated with a standard concentration of H₂O₂ (3%H₂O₂ CHIKV) for 7 hours at room temperature. Following treatment,antigen was tested with a CHIKV-specific sandwich ELISA comprised of twoneutralizing monoclonal antibodies specific for the E1 and E2 structuralproteins. ELISA values are expressed as a percentage of live viruscontrols.

In FIG. 2B, H₂O₂-treated CHIKV (3% H₂O₂ CHIKV) was tested and foundnegative for residual live virus, formulated with 0.1% alum, and used toimmunize adult BALB/c mice (n=8) on days 0 and 28. Control mice (Mock,n=3) were immunized on the same schedule with alum in diluent. Two weeksfollowing the final immunization peripheral blood was collected,processed for serum and pooled for each group. Pooled serum was testedusing a standard CHIKV 50% plaque reduction neutralization assay(PRNT₅₀). Samples from the 3% H₂O₂-CHIKV and mock vaccinated groups wereseronegative, with a PRNT₅₀ titer of less than 10, as indicated by thedashed line. For comparison, a group of C57BL/6 mice (n=5) immunizedwith live CHIKV by the intradermal footpad route (1,000 PFU ofCHIKV-SL15649) are shown (left-most bar graph of FIG. 2B), withneutralizing titers tested 36 days following infection. The limit ofdetection (LOD) is indicated by the dashed line.

EXAMPLE 2 Dual Oxidation-Based Microbial Inactivation was Found byApplicants to Have a Fundamentally Different Mechanism Compared WithSimple Oxidation With H₂O₂ Alone, Thereby Discouraging the Potential useof Dual Oxidation-Based Microbial Inactivation for the Development ofAdvanced Efficacious Vaccine Antigens

While Fenton-type reactions have only been used for killing pathogens,and have not been used or suggested for using in the development ofvaccines, such reactions were nonetheless tested for the potential toinactivate microbial pathogens for purpose of vaccine production. Theinitial inactivation data was surprising and unexpected, because incontrast to H₂O₂, it was found that the total protein concentration ofthe solution during the inactivation procedure impacts H₂O₂/CuCl₂dual-oxidation inactivation kinetics. This H₂O₂/CuCl₂ system result wasunexpected because protein concentration had been previously shown tohave no impact on viral inactivation using Applicants' standard H₂O₂approach. However, as shown in FIGS. 1A and 1B for DENV2, proteinconcentration had a substantial impact in viral inactivation kinetics,with higher protein levels leading to slower inactivation of the virus.

Specifically, FIGS. 1A and 1B show that the kinetics of virusinactivation using the H₂O₂/CuCl₂ dual oxidation system is proteinconcentration-dependent, whereas standard H₂O₂-based virus inactivationis protein concentration-independent. In FIG. 1A, purified DENV2 wastreated with either 3% H₂O₂, or in FIG. 1B with 0.01% H₂O₂ and 1 μMCuCl₂ at room temperature, with increasing concentrations of total viralprotein as indicated. Samples were removed at pre-specified time pointsand assessed for viral titers using a standard plaque forming unit (PFU)assay. The limit of detection (LOD) is indicated by the dashed line.

The dependence on total protein concentration of the solution during thedual inactivation procedure was unexpected, indicating that afundamentally different mechanism was involved compared to H₂O₂ alone,and thus the efficacy/use of a dual oxidation-based inactivationprocedure for effective vaccine production was questionable andunpredictable in view of Applicants' prior simple oxidation basedmethods (e.g., with H₂O₂ alone) (e.g., U.S. Pat. Nos. 8,124,397 and8,716,000).

EXAMPLE 3 A Dual Oxidizing Fenton-Type Oxidation System was Used toProvide Efficient Inactivation While Improving the Maintenance ofCHIKV-Specific Neutralizing Epitopes

FIG. 3 shows that the use of a dual oxidizing Fenton-type oxidationsystem provides efficient inactivation while improving the maintenanceof CHIKV-specific neutralizing epitopes.

In FIG. 3A, purified CHIKV was treated with increasing concentrations ofH₂O₂ alone.

In FIG. 3B, purified CHIKV was treated with CuCl₂ alone. In FIG. 3C,purified CHIKV was treated with CuCl₂ (10 μM) with increasingconcentrations of H₂O₂ to achieve a dual oxidizing Fenton-type system.Antigen treatments were allowed to proceed for 20 hours at roomtemperature.

Following treatments, antigen was tested with a CHIKV-specific sandwichELISA comprised of two neutralizing monoclonal antibodies specific forthe E1 and E2 structural proteins. ELISA values are expressed as apercentage of live virus controls. Following treatment, material wasalso tested for live virus using a standard plaque forming unit (PFU)assay. Resulting virus titers (PFU/mL) are indicated for each condition.Increasing concentrations of either decontamination reagent (FIGS. 3Aand 3B) led to enhanced inactivation, but at the expense ofsignificantly decreased antigenicity. Surprisingly, by contrast, usingthe combined H₂O₂/CuCl₂ system, an optimal inactivation condition wasidentified that fully maintained antigenicity while leading to completeviral inactivation (FIG. 3C). Successful conditions that demonstrated nodetectable live virus (<50 PFU/mL) are indicated by an asterisk. Notethat only the optimal conditions of 10 μM CuCl₂ and 0.002% H₂O₂ achieved≥90% retained antigenicity (indicated by the dashed line) while alsodemonstrating no detectable live virus.

EXAMPLE 4 CuCl₂/H₂O₂-CHIKV Vaccination Induced Rapid NeutralizingAntibody Responses, and Protected Against CHIKV-Associated Pathology

To assess the immunogenicity of the H₂O₂/CuCl₂-treated CHIKV candidate,vaccine antigen was formulated with alum adjuvant and used to immunizemice at several dose levels (10 or 40 μg per animal). As shown in FIG.4, vaccination generated rapid and robust neutralizing antibody titers,in stark contrast to the conventional H₂O₂ approach (FIG. 2). As a finaltest of vaccine efficacy, immunized mice were challenged with wild-typeCHIKV, and demonstrated full protection against arthritic disease (FIG.5).

FIG. 4 shows that CuCl₂/H₂O₂-CHIKV vaccination induced rapidneutralizing antibody responses. Specifically, an optimizedCuCl₂/H₂O₂-CHIKV vaccine was formulated with 0.1% alum at a 10 μg or 40μg dose with a primary dose given at day 0 and a booster dose at day 14(shown by arrows). Serum samples were collected at the indicated timepoints and assayed for CHIKV-specific neutralizing activity using astandard plaque reduction neutralization titer assay (PRNT₅₀).Neutralizing titers for the 10 μg group end on day 20 post-primaryvaccination because this is the last time point before the animals werechallenged with CHIKV on day 21. Group averages (±SEM) are shown foreach time point. The limit of detection (LOD) for this study isindicated by the dashed line. Naive, unvaccinated controls were alsotested and found to be below the LOD.

FIGS. 5A and 5B show that CuCl₂/H₂O₂-CHIKV vaccination induced rapidneutralizing antibody responses, and protected against CHIKV-associatedpathology. Specifically, the CuCl₂/H₂O₂-CHIKV vaccine was formulatedwith alum at a 10 μg or 40 μg dose with a primary immunization given atday 0 and a booster dose administered at day 14 in adult C57BL/6 mice(n=5 per group) or mock vaccinated controls (alum only). Mice werechallenged in the right footpad with 1,000 PFU of CHIKV-SL15649, avirulent strain of CHIKV, at either 32 days (40 μg group) or 21 days (10μg group) after primary vaccination. CHIKV-associated foot swelling wasmeasured with calipers for 14 days in mice vaccinated with (FIG. 5A) a40 μg dose or (FIG. 5B) a 10 μg dose. Significant differences areindicated by asterisks (Student's t-test, P<0.05).

CuCl₂/H₂O₂-CHIKV vaccination generated rapid and robust neutralizingantibody titers (FIG. 4), and demonstrated full protection againstarthritic disease (FIG. 5).

EXAMPLE 5 H₂O₂/CuCl₂-Based Oxidation was Used to Develop an EffectiveInactivated YFV Vaccine

Based on the encouraging results demonstrated with CHIKV, a modelalphavirus, the utility of the system for flaviviruses such as YFV wasexplored. Preliminary analysis suggested that a concentration of 0.002%H₂O₂ and 1 μM CuCl₂ represented a functional balance betweenantigenicity and rapid virus inactivation (FIG. 6A).

Using a further optimized condition of 0.010% H₂O₂ and 1 μM CuCl₂ (toensure full inactivation) vaccine material was produced for YFV and usedto immunize adult BALB/c mice. Following vaccination, all animalsdemonstrated measurable neutralizing titers with an average neutralizingtiter of 240, compared to a neutralizing titer of less than 40 foranimals immunized with YFV vaccine prepared using H₂O₂ alone (FIG. 6B).These differences in immunogenicity after vaccination could beanticipated based on the severe damage to neutralizing epitopes (i.e.,antigenicity) observed when YFV was treated with 3% H₂O₂ for 20 hours.

FIGS. 6A and 6B show that H₂O₂/CuCl₂-based oxidation was successfullyused in the development of an inactivated YFV vaccine, and demonstratingenhanced retention of antibody binding to neutralizing epitopes(antigenicity) and improved immunogenicity after vaccination.

Specifically, as shown in FIG. 6A, purified YFV was treated with theindicated conditions for 20 hours at room temperature. Followingtreatment, antigen was tested using a YFV-specific sandwich ELISAcomprised of a neutralizing monoclonal antibody specific for theenvelope structural protein. ELISA values are expressed as a percentageof the live virus control. Following treatment, material was also testedfor live YFV using a standard plaque forming unit (PFU) assay. Resultingvirus titers (PFU/mL) are indicated for each condition. Successfulconditions that demonstrated no detectable live virus are indicated byan asterisk.

Specifically, as shown in FIG. 6B, immunization of mice with thestandard H₂O₂-based inactivated YFV (3% H₂O₂ for 7 hours) was comparedto an optimized H₂O₂/CuCl₂ condition (0.01% H₂O₂, 1 μM CuCl₂, 20 hoursat room temperature). Following inactivation, vaccine preparations weretested and found negative for live virus. Each vaccine was formulatedwith alum at a 5 μg (3% H₂O₂) or 10 μg (0.01% H₂O₂, 1 μM CuCl₂) dosewith a primary immunization given at day 0 and booster dosesadministered at days 14 and 25 in adult BALB/c mice (n=5 per group).Animals were tested for neutralizing antibody titers on day 42. Thelimit of detection (LOD) is indicated by the dashed line.

H₂O₂/CuCl₂-based oxidation, therefore, was successfully used in thedevelopment of an inactivated YFV vaccine, and demonstrating enhancedretention of antibody binding to neutralizing epitopes (antigenicity)and improved immunogenicity after vaccination.

EXAMPLE 6 H₂O₂/CuCl₂-Based Oxidation was Successfully Used in theDevelopment of an Inactivated DENV Vaccine

Based on the encouraging results demonstrated with YFV, another modelflavivirus, dengue 3 (DENV3) was tested in the H₂O₂/CuCl₂ system.

As with YFV, initial tests indicated that a concentration of 0.002% H₂O₂and 1 μM CuCl₂ represented an optimal approach for maintaining highantigenicity while also providing complete virus inactivation (FIG. 7).

Specifically, FIG. 7 shows that use of a dual oxidizing Fenton-typeoxidation system demonstrated enhanced inactivation while maintainingdengue virus 3-specific neutralizing epitopes. Purified dengue virus 3(DENV3) was treated with the indicated conditions for 20 hours at roomtemperature. Following treatment, antigen was tested with aDENV-specific sandwich ELISA comprised of two neutralizing monoclonalantibodies specific for the envelope structural protein. ELISA valuesare expressed as a percentage of the live virus control. Followingtreatment, material was also tested for live DENV3 using a standardplaque forming unit (PFU) assay. Resulting virus titers (PFU/mL) areindicated for each condition. Successful conditions that demonstrated nodetectable live virus (<50 PFU/mL) are indicated by an asterisk. Notethat only the optimal conditions of 1 μM CuCl₂ and 0.002% H₂O₂ retainedhigh antigenicity while also demonstrating no detectable live virus

Using these preliminary H₂O₂/CuCl₂ inactivation conditions, vaccine lotsof each DENV serotype were produced, formulated into a tetravalentdengue vaccine adjuvanted with 0.10% aluminum hydroxide, and used toimmunize adult rhesus macaques. Following a single booster immunization,all monkeys seroconverted (NT₅₀≥10), with the H₂O₂/CuCl₂ inactivationapproach demonstrating an improvement in neutralizing antibody responsesfor 3 out of 4 dengue virus serotypes and an average 8-fold increase ingeometric mean titers when compared to inactivation with H₂O₂ alone(FIG. 8).

Specifically, FIG. 8 shows that The H₂O₂/CuCl₂ dual-oxidation systemenhanced in vivo immunogenicity to a tetravalent DENV vaccine in rhesusmacaques. Purified DENV was treated with either 3% H₂O₂ (7 hours, roomtemperature) or H₂O₂/CuCl₂ (0.002% H₂O₂ and 1 μM CuCl₂ for 20 hours,room temperature). Full inactivation was confirmed through standardplaque assay and co-culture. Vaccine antigens were blended at equalconcentrations (1 μg per serotype for 3% H₂O₂, or 2 μg per serotype forH₂O₂/CuCl₂) to and formulated with 0.1% alum. Adult rhesus macaques (n=4per group) were immunized intramuscularly at day 0 and day 28, withneutralization titers (NT₅₀) measured at 1-month following boosterimmunization. The limit of detection (LOD) is indicated by the dashedline.

There was a small difference in antigen dose (1 μg/serotype vs. 2μg/serotype) in these studies and so the experiment was repeated in micethat were vaccinated with the same dose of tetravalent dengue vaccineantigen (FIG. 9).

Specifically, FIG. 9 shows that The H₂O₂/CuCl₂ dual-oxidation systemenhances in vivo immunogenicity to a tetravalent DENV vaccine in mice.Purified DENV was treated with either 3% H₂O₂ (7 hours, roomtemperature) or H₂O₂/CuCl₂ (0.002% H₂O₂ and 1 μM CuCl₂ for 20 hours,room temperature). Full inactivation was confirmed through standardplaque assay and co-culture. Vaccine antigens were blended at equalconcentrations (2 μg per serotype) and formulated with 0.1% alum. AdultBALB/c mice (n=4-5 per group) were immunized subcutaneously at days 0,14 and day 28, with neutralization titers (NT₅₀) measured at two-weeksfollowing the final immunization. The limit of detection (LOD) isindicated by the dashed line.

In these experiments, the dual oxidation approach of H₂O₂/CuCl₂inactivation was more immunogenic than 3% H₂O₂ for all 4 dengue virusserotypes and resulted in an 8-fold to >800-fold improvement inneutralizing antibody titers.

EXAMPLE 7 CuCl₂/H₂O₂-Based Oxidation Demonstrated Improved AntigenicityWith Influenza Virus

Given the positive results observed across two virus families(Togaviridae and Flaviviridae), an additional virus family was chosen totest using this new inactivation platform.

As shown in this working example, inactivation of Influenza A virus(family Orthomyxoviridae) was tested using a standard 3% H₂O₂ approach,ultraviolet inactivation, or the optimized CuCl₂/H₂O₂ system (0.002%H₂O₂ and 1 μM CuCl₂). To assess antigenicity, a hemagglutinationactivity (HA) titration assay was used. Influenza viruses naturallyagglutinate red blood cells, and maintenance of this activity throughoutinactivation is considered key to the immunogenicity of the finalvaccine product. As shown in FIG. 10, Applicants' CuCl₂/H₂O₂ systemmaintained HA titers similar to that observed for live, untreatedantigen.

Specifically, FIG. 10 shows that CuCl₂/H₂O₂-based virus inactivationmaintained influenza hemagglutination activity better than H₂O₂ alone.Purified influenza A/PR/8/34 (H1N1) was inactivated with H₂O₂ (3% for 2hours, room temperature) CuCl₂/H₂O₂ (1 μM CuCl₂, 0.002% H₂O₂ for 20hours, room temperature), ultraviolet light (UV, 10 joules) or leftuntreated (Live). Following inactivation, antigen preparations weredirectly tested for hemagglutination (HA) activity. Antigen preparationswere scored by the lowest antigen concentration that still demonstratedfull HA activity, and the reciprocal of this concentration was graphed.CuCl₂/H₂O₂ maintained protein function (i.e., hemagglutination activity)at levels that were indistinguishable from live influenza.

By comparison, UV inactivation reduced HA activity to a negligiblelevel. The in vivo consequence of this HA destruction can be seen inFIG. 11, with the CuCl₂/H₂O₂ inducing robust protective serum antibodyhemagglutinin inhibition (HAI) titers, while UV-treated antigen inducedno functional antibodies in mice and minimal protection against lethalchallenge.

Specifically, FIG. 11 shows that CuCl₂/H₂O₂ inactivated influenzainduced robust hemagglutination inhibition titers and protected againstlethal challenge. Purified influenza A/PR/8/34 (H1N1) was inactivatedwith H₂O₂ (3% for 2 hours, room temperature), CuCl₂/H₂O₂ (1 μM CuCl₂,0.002% H₂O₂ for 20 hours, room temperature) or ultraviolet light (UV, 10joules), with complete inactivation confirmed through focus formingassay viability testing. Following inactivation, antigen preparationswere normalized by protein content and formulated with 0.10% aluminumhydroxide. Adult female BALB/c mice were immunized subcutaneously with 5μg of vaccine.

FIG. 11A shows that serum influenza-specific hemagglutinin inhibition(HAI) titers were determined for animals at two months post-vaccination.Results from unvaccinated control mice are shown for comparison. Thelimit of detection (LOD) for the assay is indicated by the dashed line.

FIG. 11B shows that at two months post-immunization, mice werechallenged intranasally with 6×10⁴ EID₅₀ of live influenza (A/PR/8/34(H1N1), 20 LD₅₀) and followed daily for changes in body weight. Anyanimals reaching ≤75% of initial starting weight were humanelyeuthanized.

Mice vaccinated with CuCl₂/H₂O₂-inactivated virus or H₂O₂-inactivatedvirus showed highly significant protection following influenzaechallenge (P=0.0031 and P=0.015, respectively). Whereas mice vaccinatedwith UV-inactivated virus demonstrated no significant protection(P=0.25).

EXAMPLE 8 Multiple Transition Metals Were Successfully Used in theDual-Oxidation Approach to Vaccine Antigen Development

Cu²⁻ (in the form of CuCl₂) was the initial metal tested in thedual-oxidation vaccine antigen development studies described for CHIKV,DENY, YFV and influenza virus. However, as described above, Applicantsdetermined that other metals also have the potential to function in asimilar manner.

As shown in this example using DENV3 as a model virus, inactivationstudies consisting of CuCl₂ (Cu²⁻), FeCl₃ (Fe³⁺) or CsCl (Cs⁺) anddilutions of H₂O₂ were tested for their potential in the development ofvaccine antigen.

As shown in FIGS. 12A-C, all three metals provided conditions thatmaintained high levels of antigenicity while demonstrating completevirus inactivation.

Specifically, FIGS. 12A-C show a comparison of redox-active metals fordual oxidation-based virus inactivation. Purified DENV3 was treated witha range of H₂O₂ concentrations as indicated (20 hours, room temperature)in the presence of increasing concentrations of CuCl₂ (FIG. 12A), FeCl₃(FIG. 12B) and CsCl (FIG. 12C). Following treatment, the maintenance ofneutralizing antibody binding sites (i.e., antigenicity) was measuredusing a DENV-specific sandwich ELISA comprised of two neutralizingmonoclonal antibodies specific for the DENV envelope protein. ELISAvalues are expressed as a percentage of the live virus control.Following treatment, material was also tested for live DENV3 using astandard plaque forming unit (PFU) assay. Successful conditions thatdemonstrated no detectable live virus (<50 PFU/mL) are indicated by anasterisk (and where “N.T.” is not tested).

All three metals provided conditions that maintained high levels ofantigenicity while demonstrating complete virus inactivation.

EXAMPLE 9 Combinations of Transition Metals Demonstrated Synergy in theDual-Oxidation Vaccine System

As shown above in FIG. 12 and working example 8, different metals can beused in combination to enhance H₂O₂ inactivation of viruses.

As shown in this working example, to investigate potential synergisticeffects, DENV3 model virus was inactivated with combinations of CuCl₂(Cu²⁺) and FeCl₃ (Fe³⁺) at a set amount of H₂O₂ (0.01%). A number ofCuCl₂/FeCl₃ conditions provided full inactivation while maintaining goodantigenicity, demonstrating that using multiple metals in the sameinactivation condition is feasible (FIG. 13). Indeed, at CuCl₂concentrations of 0.05 μM and 0.10 μM, increasing FeCl₃ concentrationsenhanced antigenicity, indicating synergy with these two metals.

Specifically, FIG. 13 shows that combinations of metals can achievecomplete inactivation while maintaining good antigenicity. PurifiedDENV3 was treated with H₂O₂ (0.01%) and the indicated range of CuCl₂ andFeCl₃ concentrations. Following treatment, antigen was tested with aDENV-specific sandwich ELISA comprised of two neutralizing monoclonalantibodies specific for the envelope structural protein. ELISA valuesare expressed as a percentage of the live virus control. Followingtreatment, material was also tested for live DENV3 using a standardplaque forming unit (PFU) assay. Successful conditions that demonstratedno detectable live virus (<50 PFU/mL) are indicated by an asterisk. AtCuCl₂ concentrations of 0.05 μM and 0.10 μM, increasing FeCl₃concentrations enhanced antigenicity, indicating synergy with these twometals.

EXAMPLE 10 Dual Oxidation was Used to Provide Optimized Inactivation ofCampylobacter for Improved Maintenance of Bacterial Morphology

As shown in this working example, Campylobacter are smallcorkscrew-shaped bacteria that are typically ˜0.2 μm in diameter and˜2-8 μm in length (FIG. 14A).

Following inactivation with a standard 3% H₂O₂ solution for 5 hours atroom temperature, the bacteria were substantially damaged with clearchanges in morphology, including loss of gross cellular structure andsubstantial clumping (FIG. 14B). However, upon optimization of adual-oxidation approach using 0.01% H₂O₂ and 2 uM CuCl₂, Applicantssurprisingly found that dual oxidation could completely inactivate thebacteria while maintaining excellent bacterial morphology throughout thetreatment period with microbes that remained indistinguishable from theuntreated controls (FIG. 14C).

Specifically, FIGS. 14A-14C show optimized inactivation of Campylobacterfor improved maintenance of bacterial morphology.

In FIG. 14A, C. coli was grown, purified and left untreated.

In FIG. 14B, C. coli was grown, purified and inactivated with a high butdestructive concentration of H₂O₂ (3% H₂O₂ for 5 hrs).

In FIG. 14C, C. coli was grown, purified and inactivated with 2 μM CuCl₂and 0.01% H₂O₂. Data shows samples from each condition that were appliedto slides and stained with Gram safranin.

In addition to retained structure, a critical parameter for preparing aninactivated whole-cell vaccine is to ensure complete microbeinactivation. Using the optimal conditions described above, inactivationkinetic studies were performed. As shown in FIG. 15, C. colidemonstrated rapid inactivation, with a decay rate half-life of(T_(1/2)) of ˜15 minutes.

Specifically, FIG. 15 shows that exposure to an optimized CuCl₂/H₂O₂formula results in rapid inactivation of Campylobacter. Purifiedpreparations of C. coli were treated with an optimized CuCl₂/H₂O₂formula and buffer condition, or mock inactivated (no CuCl₂/H₂O₂).Samples were taken at the indicated points and tested for viableCampylobacter. Open symbols indicate the absence of live bacteria. Thedashed line shows the limit of detection. These kinetics indicate >20logs of inactivation during the full 20-hr inactivation period. Based onthe bacterial titers in our pilot manufacturing lots (˜10⁹ CFU/mL) thislevel of inactivation provides a high safety margin during themanufacturing process (up to 100 million-fold theoretical excessinactivation) while still maintaining overall bacterial structure (FIG.14C).

EXAMPLE 11 Dual Oxidation-Campylobacter Vaccination Provides ProtectiveImmunity in Rhesus Mmacaques

As shown in this working example, Applicants determined vaccine efficacythrough the monitoring of Campylobacter culture-confirmed entericdisease rates in 60 CuCl₂/H₂O₂-C. coli-immunized rhesus macaques ascompared to unvaccinated control animals.

For this study, animals were vaccinated intramuscularly with theCuCl₂/H₂O₂-C. coli vaccine candidate (inactivated using 0.01% H₂O₂ and 2μM CuCl₂), with a booster dose administered 6-months later. Vaccinatedgroups were selected based on prior disease history, with preferencegiven to groups that had historically high incidence rates ofCampylobacter infection. This approach provided increased robustness inevaluating protective efficacy. All adults/juveniles (n=59) received a40-μg alum-adjuvanted dose, with 2 small infants (<2 Kg body weight)receiving a half-dose (20-μg). According to protocol, any animaldiagnosed with Campylobacter-associated diarrhea during the first 14days after vaccination would be excluded since vaccine-mediatedprotection would be unlikely to occur during this early period. Oneadult animal was excluded from the study due to Campylobacter-associateddiarrhea on the day after vaccination. Serum samples were collected fromall remaining vaccinated animals (n=59) at day 0 and at 6 months afterprimary vaccination at which time the animals received a booster dose ofvaccine.

Following primary vaccination, we observed a significant increase inCampylobacter-specific serum antibody titers (FIG. 16A, P<0.001) inaddition to protection against Campylobacter-associated diarrhealdisease in comparison with prior years within the same shelter group(FIG. 16B, P=0.038) or in comparison with other shelter groups duringthe 2015 Campylobacter season (FIG. 16C, P=0.020). The health of NHP ismonitored daily and cases of diarrheal disease are documented in asearchable central database. Diarrhea incidence was monitored in thevaccinated cohort and compared to approximately 1,000 unvaccinatedcontrol animals in other similar shelter groups. Fecal samples werecollected from any animal experiencing a diarrheal episode and testedfor C. coli, C. jejuni, and Shigella spp. since these represent the mainenteric pathogens associated with diarrhea among the animals.

Specifically, FIGS. 16A-16C show that dual oxidation-C. coli isimmunogenic and protects RM against naturally acquired Campylobacterinfection.

In FIG. 16A, serum samples were collected from animals just prior tovaccination, or 6 months following primary immunization and assayed forCampylobacter-specific antibody responses using an optimized, whole-cellELISA, with all serum samples pre-adsorbed against Shigella (agram-negative enteric bacteria) to reduce non-specific binding.Significance testing was performed using a paired student's t-test.

Subsequent to vaccination, animals were followed for 8 months for C.coli-associated diarrhea, and compared (FIG. 16B) to prior year diarrhearates within the same shelter, or compared (FIG. 16C) to the rates ofdiarrheal incidence in other concurrent shelters (˜1,000 controlanimals) monitored in 2015. Black arrows indicate the time of boostervaccination.

Interim analysis at 6 months after primary vaccination demonstrated nocases of C. coli or C. jejuni-associated diarrhea in the vaccinatedgroup versus 76 cases of Campylobacter-associated diarrhea among theunvaccinated animals, representing a statistically significantprotective effect against Campylobacter culture-positive diarrhealdisease (P=0.035) after a single vaccination.

Since nearly all human vaccines require at least two doses for optimalprotective efficacy and the durability of immunological memory is oftenimproved following booster vaccination, a conservative approach wasfollowed by administering a booster vaccination at the 6 month timepoint followed by continued monitoring of the incidence of diarrhealdisease among the NHP. At 250 days after primary vaccination, more casesof Campylobacter-associated enteric disease had continued to accrueamong the unvaccinated population (reaching 8.7% or a total of 92animals) whereas none of the animals (0/59) in the vaccinated cohortshowed signs of disease and the statistical significance between the twogroups increased to P=0.020.

EXAMPLE 12 Methisazone Enhanced the Rate of Both Single and DualOxidation-Based Virus Inactivation

As shown in this working example, Applicants determined that methisazoneenhanced the rate of both single and dual oxidation-based virusinactivation. As shown in FIGS. 17A-C, the addition of methisazone wasable to substantially increase the rate of dual-oxidation-basedinactivation for vaccinia virus (VV, DNA genome) as well as dengue virusserotype 4 (DENV4, RNA genome) and chikungunya virus (CHIKV, RNAgenome).

Further, while methisazone alone had a minimal impact on virusinactivation (FIGS. 17B & 17C), methisazone and H₂O₂ together (even inthe absence of copper) demonstrated a synergistic enhancement for virusinactivation.

Specifically, FIGS. 17A, 17B, and 17C show, according to particularaspects, that methisazone enhanced the rate of both single and dualoxidation-based virus inactivation. (A) Vaccinia virus (PBS, pH=7.5),(B) dengue virus serotype 4 (DENV4, in 110 mM NaCl, 150 mM NaPO₄[pH=7.5], 2% D-sorbitol) and (C) Chikungunya virus (CHIKV, in PBSsupplemented with 150 mM NaPO₄ [pH=7.5]), were each treated withinactivation reagents as indicated in the figure. Concentrations for thedifferent components were as follows: H₂O₂=0.004% (CHIKV) or 0.002%(DENV4 and VV); CuCl₂=1 μM (all viruses), methisazone (MZ)=10 μM (allviruses). The dotted line indicates the limit of detection (LOD).

EXAMPLE 13 Methisazone Enhanced the Rate of Dual Oxidation-BasedBacterial Inactivation

As shown in this working example, Applicants determined that methisazoneenhanced the rate of dual oxidation-based bacterial inactivation. Theresults of working Example 12 were extended to bacteria (FIGS. 18A-C)where again the addition of methisazone to the dual-oxidation approach(e.g., H₂O₂/CuCl₂) substantially enhanced inactivation rates forCampylobacter coli (an exemplary gram-negative bacteria), Listeriamonocytogenes (an exemplary gram-positive bacteria) and Shigelladysenteriae (an exemplary gram-negative bacteria).

Specifically, FIGS. 18A, 18B, and 18C show, according to particularaspects, that methisazone enhanced the rate of dual oxidation-basedbacterial inactivation. (A) Campylobacter coli (B) Listeriamonocytogenes and (C) Shigella dysenteriae were buffer exchanged into 10mM NaCl, 150 mM NaPO₄ [pH=7.5] and 2% D-sorbitol and treated withinactivation components as indicated in each panel. Viabilitypost-inactivation, as determined through colony forming units per mL(CFU/mL), was followed over time. Concentrations of inactivationcomponents were optimized for each type of bacteria as follows: C. coli:H₂O₂=0.01%, CuCl₂=2 μM, methisazone (MZ)=20 μM; L. monocytogenes:H₂O₂=0.10%, CuCl₂=10 μM, methisazone (MZ)=100 μM. S. dysenteriae:H₂O₂=0.10%, CuCl₂=10 μM, MZ=100 μM; Open symbols represent conditionswithout MZ, while closed symbols indicate the addition of MZ. The limitof detection was 10 CFU/mL.

EXAMPLE 14 Methisazone Enhanced Inactivation Rates While MaintainingAntigenicity During Dual Oxidation-Based Viral Inactivation

As shown in this working example, Applicants determined that methisazoneenhanced inactivation rates while maintaining antigenicity during dualoxidation-based virus inactivation. To assess the impact of methisazoneon antigenicity during inactivation, the exemplary model viruses CHIKVand DENV4 were treated with multiple inactivation approaches: highconcentration H₂O₂ (single oxidation system), dual-oxidation (asdescribed herein), or dual-oxidation with methisazone. As shown by theELISA data in FIGS. 19A (Chikungunya virus (CHIKV)) and 19B (denguevirus serotype 4 (DENV4)), the addition of methisazone to thedual-oxidation approach maintained or significantly improvedantigenicity by reducing damage to neutralizing epitopes, whileincreasing the rate of inactivation by approximately 10- to 20-fold.

Specifically, FIGS. 19A and 19B show, according to particular aspects,that methisazone enhanced inactivation rates while maintainingantigenicity during dual oxidation-based virus inactivation. Chikungunyavirus (CHIKV, in PBS supplemented with 150 mM NaPO₄ [pH=7.5]) and denguevirus serotype 4 (DENV4, in 110 mM NaCl, 150 mM NaPO₄ [pH=7.5], 2%D-sorbitol) were each treated for 20 hours at room temperature with theinactivation components indicated in the figure. Following virustreatment, antigen retention was tested with either (A) a CHIKV-specificsandwich ELISA comprised of two neutralizing monoclonal antibodiesspecific for the E1 and E2 structural proteins or (B) a DENV-specificsandwich ELISA comprised of two neutralizing monoclonal antibodiesspecific for the envelope structural protein. ELISA values indicateretained neutralizing epitopes and are expressed as a percentage of livevirus controls. Both viruses were also treated with 3% H₂O₂ to show lossof neutralizing epitopes by a damaging inactivation approach.Inactivation half-lives for each condition are shown.

EXAMPLE 15

Chemical Analogs of Methisazone, or Methisazone FunctionalGroups/Substructures or Combinations Thereof Enhanced Inactivation andMaintenance of Antigenicity During Dual Oxidation-Based ViralInactivation)

As shown in this working example, Applicants determined that chemicalanalogs of methisazone, or methisazone functional groups/substructuresor combinations thereof, enhanced inactivation and maintenance ofantigenicity during dual oxidation-based viral inactivation.

As mentioned above, methisazone is a compound originally developed as anin vivo antiviral agent. We tested several related compounds todetermine if they provided similar enhancements to pathogen inactivationfor vaccine development (FIGS. 20A-C). As shown with the exemplary modelvirus DENV4, several of these compounds, such as isatinβ-thiosemicarbazone and N-propylisatin β-thiosemicarbazone, demonstratedresults similar to methisazone including enhanced rates of inactivationwhile maintaining superior antigenicity in the dual-oxidation system.Interestingly, when using just the thiosemicarbazide moiety, we stillobserved enhancement of inactivation and superior antigenicity, whereasisatin or semicarbazide do not appear to increase the rate ofinactivation, but still demonstrate protection of protein antigens fromoxidative damage during inactivation. To explore if the separate majorcomponents (functional groups/substructures) of methisazone-relatedcompounds could be combined in order to recapitulate optimalinactivation, we tested mixtures of isatin+thiosemicarbazide orisatin+semicarbazide. While isatin+semicarbazide still demonstratedantigen protection, there was no enhancement of virus inactivation. Bycontrast, isatin+thiosemicarbazide resulted in both rapid inactivation(more rapid than either component alone) as well as greatly increasedantigenicity.

Specifically, FIGS. 20A, 20B, and 20C show, according to particularaspects, that chemical analogs of methisazone, or methisazone functionalgroups/substructures or combinations thereof, enhanced inactivation andmaintenance of antigenicity during dual oxidation-based viralinactivation. (A) Related chemical compounds of the isatinβ-thiosemicarbazone class are shown. (B) Dengue virus serotype 4 (DENV4,in 110 mM NaCl, 150 mM NaPO₄ [pH=7.5], 2% D-sorbitol) was treated withdual oxidation components as indicated in each panel (H₂O₂=0.01%,CuCl₂=1 μM) in the absence or presence of different MZ-like compounds,with each compound used at a concentration of 10 μM. To assessinactivation, viable virus was tested by plaque assay at 1 hrpost-inactivation. The dotted line indicates the limit of detection. (C)To quantitate antigenicity, a DENY-specific sandwich ELISA comprised oftwo neutralizing monoclonal antibodies specific for the envelopestructural protein was performed at 20 hrs post-inactivation. ELISAvalues indicate retained neutralizing epitopes and are expressed as apercentage of live virus controls.

EXAMPLE 16 Increasing Levels of Methisazone Relative to the TransitionMetal Component of the Dual Oxidation System Improved the Antigenicityand Inactivation Profile of the Dual Oxidation System

As shown in this working example, Applicants determined that increasinglevels of methisazone relative to the transition metal component of thedual oxidation system improved the antigenicity and inactivation profileof the dual oxidation system. We examined the impact of relativeconcentrations of methisazone and the transition metal in thedual-oxidation system (FIG. 21). We found that increasing methisazoneconcentrations relative to the transition metal demonstrated concomitantimprovements in both retained antigenicity and increased virusinactivation rates, with a preferred molar ratio of 10:1 (methisazone:transition metal).

Specifically, FIG. 21 shows, according to particular aspects, thatincreasing levels of methisazone relative to the transition metalcomponent of the dual oxidation system improved the antigenicity andinactivation profile of the dual oxidation system. Chikungunya virus(CHIKV, in PBS supplemented with 150 mM NaPO₄ [pH=7.5]) was treated withH₂O₂ (0.02%) and CuCl₂ (1 μM) at room temperature in the presence ofdecreasing concentrations of methisazone. Following treatment, virus wastested by plaque assay at 1 hr to assess inactivation, and tested forretained antigenicity at 20 hrs using a CHIKV-specific sandwich ELISAcomprised of two neutralizing monoclonal antibodies specific for the E1and E2 structural proteins. The limit of detection for the plaque assayis indicated by the dotted line.

References supporting the working examples and incorporated by referenceherein for their respective teachings:

Sagripanti, J. L., L. B. Routson, and C. D. Lytle, Virus inactivation bycopper or iron ions alone and in the presence of peroxide. Appl EnvironMicrobiol, 1993. 59(12): p. 4374-6.

Nieto-Juarez, J. I., et al., Inactivation of MS2 coliphage in Fenton andFenton-like systems: role of transition metals, hydrogen peroxide andsunlight. Environ Sci Technol, 2010. 44(9): p. 3351-6.

Barbusiński, K., Fenton Reaction-Controversy concerning the chemistry.Ecological Chemistry and Engineering, 2009. 16(3): p. 347-358.

Sagripanti, J. L., Metal-based formulations with high microbicidalactivity. Appl Environ Microbiol, 1992. 58(9): p. 3157-62.

McClatchey, K. D., Clinical laboratory medicine. 2nd ed. 2002,Philadelphia: Lippincott Wiliams & Wilkins. xiv, 1693 p.

Lippincott Williams & Wilkins., Nursing. Deciphering diagnostic tests.Nursing. 2008, Philadelphia, Pa.: Wolters Kluwer/Lippincott Williams &Wilkins. vii, 664 p.

Sagripanti, J. L., et al., Mechanism of copper-mediated inactivation ofherpes simplex virus. Antimicrob Agents Chemother, 1997. 41(4): p.812-7.

Sagripanti, J. L., P. L. Goering, and A. Lamanna, Interaction of copperwith DNA and antagonism by other metals. Toxicol Appl Pharmacol, 1991.110(3): p. 477-85.

Toyokuni, S. and J. L. Sagripanti, Association between8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated bycopper and iron, in Free Radic Biol Med. 1996: United States. p. 859-64.

Nguyen, T.T., et al., Microbial inactivation by cupric ion incombination with H2O2: role of reactive oxidants. Environ Sci Technol,2013. 47(23): p. 13661-7.

Thompson R L, Minton S A, Jr., Officer J E, Hitchings G H. Effect ofheterocyclic and other thiosemicarbazones on vaccinia infection in themouse. J Immunol. 1953;70:229-34.

Bauer D J. The antiviral and synergic actions of isatinthiosemicarbazone and certain phenoxypyrimidines in vaccinia infectionin mice. Br J Exp Pathol. 1955;36:105-14.

Bauer D J. Clinical experience with the antiviral drug marboran(1-methylisatin 3-thiosemicarbazone). Ann N Y Acad Sci. 1965;130:110-7.

Bauer D J, Stvincent L, Kempe C H, Downie A W. Prophylactic Treatment ofSmall Pox Contacts with N-Methylisatin Beta-Thiosemicarbazone (Compound33t57, Marboran). Lancet. 1963;2:494-6.

Fox M P, Bopp L H, Pfau C J. Contact inactivation of RNA and DNA virusesby N-methyl isatin beta-thiosemicarbazone and CuSO4. Ann N Y Acad Sci.1977; 284:533-43.

Logan J C, Fox M P, Morgan J H, Makohon A M, Pfau C J. Arenavirusinactivation on contact with N-substituted isatinbeta-thiosemicarbazones and certain cations. J Gen Virol. 1975;28:271-83.

Mikelens P E, Woodson B A, Levinson W E. Association of nucleic acidswith complexes of N-methyl isatin-beta-thiosemicarbazone and copper.Biochem Pharmacol. 1976; 25:821-7.

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1. A method for producing an immunogenic vaccine composition comprisingan inactivated pathogen, the method comprising: contacting a pathogenwith a Fenton reagent, comprising hydrogen peroxide in combination witha transition metal, in an amount and for a time-period sufficient forthe agent to render the pathogen noninfectious while retaining pathogenimmunogenicity.
 2. The method of claim 1, further comprising verifyingimmunogenicity of the noninfectious pathogen using pathogen-specificantibody, B cell or T cell immunoassays, agglutination assays, or othersuitable assays, wherein producing an immunogenic vaccine compositioncomprising an inactivated pathogen is afforded.
 3. The method of claim1, wherein the Fenton reagent comprises hydrogen peroxide in combinationwith at least one transition metal ion selected from the groupconsisting of Cu, Fe, and Cs.
 4. The method of claim 1, wherein amixture of different transition metal ions are used in combination withhydrogen peroxide.
 5. The method of claim 1, wherein the pathogen genomecomprises RNA or DNA.
 6. The method of claim 5, wherein the pathogen isa virus, or a bacterium.
 7. The method of claim 6, wherein the pathogenis a virus.
 8. The method of claim 7, wherein the virus is from FamilyTogaviridae, Flaviviridae, Poxviridae or Orthomyxoviridae.
 9. The methodof claim 7, wherein the virus is from Family: Mgaviridae, Genus:Alphavirus), Family: Flaviviridae, Genus: Flavivirus), Family:Poxviridae, Genus Orthopoxvirus, or Family: Orthomyxoviridae, Genus:Influenzavirus.
 10. The method of claim 9, wherein the virus ischikungunya virus (CHIKV, Family: Togaviridae, Genus: Alphavirus),dengue virus serotypes 1-4 and yellow fever virus (DENY 1-4, YFV,Family: Flaviviridae, Genus: Flavivirus vaccinia virus (VV, Family:Poxviridae, Genus: Orthopoxvirus) or influenza virus (Family:Orthomyxoviridae, Genus: Influenzavirus.
 11. The method of claim 6,wherein the pathogen is a bacterium.
 12. The method of claim 11, whereinthe bacterium is Campylobacter.
 13. The method of claim 12, wherein theCampylobacter is C. coli or C. jejuni.
 14. The method of claim 11,wherein the bacterium is Shigella spp.
 15. The method of claim 11,wherein the bacterium is Listeria spp.
 16. The method of claim 1,wherein the pathogen is isolated or purified prior to contacting withthe Fenton reagent.
 17. The method of claim 1, wherein contacting thepathogen comprises contacting the pathogen with the Fenton reagent and acompound having formula I:

wherein R₁ is independently H or lower alkyl (e.g., C1-C4 alkyl)optionally substituted with —OH; wherein R₂ is independently H, loweralkyl (e.g., C1-C2 alkyl) optionally substituted with —OH or with aryl;and wherein X is independently H or halogen; and pharmaceuticallyacceptable salts thereof.
 18. The method of claim 17, wherein X and R₂are H; and wherein R₁ is H (isatin β-thiosemicarbazone). —CH₃(N-methyl-isatin β-thiosemicarbazone (methisazone)), or propyl(N-propyl-isatin β-thiosemicarbazone).
 19. The method of claim 18,wherein R₁ is —CH₃ (N-methyl-isatin β-thiosemicarbazone (methisazone))


20. The method of any claim 1, wherein contacting the pathogen comprisescontacting the pathogen with the Fenton reagent and one or morecompounds each having one of formulas II-V:

wherein R₁ is H or lower alkyl (e.g., C1-C4) alkyl optionallysubstituted with —OH; and wherein X is independently H or halogen; andsalts, including pharmaceutically acceptable salts thereof;

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH; wherein X is independently H or halogen; andwherein R₂ is independently lower alkyl (e.g., C1-C2 alkyl) optionallysubstituted with —OH, or with aryl; and salts, includingphamiaceutically acceptable salts thereof and

wherein R₂ and R₃ are independently H, lower alkyl (e.g., C1-C2 alkyl)optionally substituted with —OH, or with aryl; and salts, includingpharmaceutically acceptable salts thereof and combinations thereof. 21.The method of claim 20, wherein X of formula II is H, and R₁ of formula(II) is H (isatin), —CH; (N-methyl-isatin), or propyl (N-propyl-isatin);wherein X, R₁ and R₂ of formula (III) are H (indole, 2,3-dione,3-hydrazone); wherein R₂ and R₃ of formula (IV) are H(thiosemicarbazide); and wherein R₂ and R₃ of formula (V) are H(semicarbazide).
 22. The method of claim 20, wherein contacting thepathogen comprises contacting the pathogen with the Fenton reagent,thiosemicarbazide and a compound having formula VI:

wherein R₁ is H or lower alkyl (e.g., C 1-C4 alkyl).
 23. The method ofclaim 22, wherein R₁ is H (isatin), —CH₃ (N-methyl-isatin), or propyl(N-propyl-isatin).
 24. The method of claim 22, wherein R₁ is H (isatin).25. An immunogenic vaccine composition having an inactivated pathogen,produced by the method of claim
 1. 26. The immunogenic vaccinecomposition of claim 25, wherein the inactivated pathogen retains one ormore predominant antigenic epitopes of the biologically active pathogensuitable to elicit a pathogen-specific antibody, B cell or T cellresponse, or to reduce infection by the pathogen, or decrease symptomsthat result from infection by the pathogen.
 27. The immunogenic vaccinecomposition of claim 25, wherein the pathogen genome comprises RNA orDNA.
 28. A method of eliciting an immune response against a pathogen,the method comprising: obtaining an immunogenic vaccine compositionhaving an inactivated pathogen, produced by the method of claim 1: andadministering the immunogenic vaccine composition to a subject, therebyeliciting in the subject an immune response against the pathogen. 29.The method of claim 28, wherein the pathogen genome comprises RNA orDNA.
 30. A method for producing an immunogenic vaccine compositioncomprising an inactivated pathogen, the method comprising: contacting apathogen with hydrogen peroxide in combination with a methisazone-typereagent selected from the group consisting of methisazone, a methisazoneanalog(s), a methisazone functional group/substructure, and combinationsthereof, in an amount and for a time-period sufficient for the agent torender the pathogen noninfectious while retaining pathogenimmunogenicity.
 31. The method of claim 30, further comprising verifyinginununogenicity of the noninfectious pathogen using pathogen-specificantibody, B cell or T cell immunoassays, agglutination assays, or othersuitable assays.
 32. The method of claim 30, wherein: the pathogengenome comprises RNA or DNA.
 33. An immunogenic vaccine compositionhaving an inactivated pathogen, produced by the method of claim
 30. 34.A method of eliciting an immune response against a pathogen, the methodcomprising: obtaining an immunogenic vaccine composition having aninactivated pathogen, produced by the method claim 33; and administeringthe immunogenic vaccine composition to a subject, thereby eliciting inthe subject an immune response against the pathogen.
 35. A method forinactivating a pathogen, the method comprising: contacting a pathogenwith hydrogen peroxide, or a Fenton reagent containing hydrogen peroxidein combination with a transition metal, and a methisazone reagent, in anamount and for a time-period sufficient to render the pathogennoninfectious.
 36. The method claim 35, wherein inactivation of thepathogen proceeds at an increased rate relative to that produced bycontacting the pathogen with either the hydrogen peroxide or Fentonreagent alone.
 37. The method of claim 35, wherein the Fenton reagentcomprises hydrogen peroxide in combination with at least one transitionmetal ion selected from the group consisting of Cu, Fe, and Cs.
 38. Themethod of claim 35, wherein a mixture of different transition metal ionsare used in combination with hydrogen peroxide.
 39. The method of claim35, wherein the pathogen genome comprises RNA or DNA.
 40. The method ofclaim 39, wherein the pathogen is a virus, or a bacterium.
 41. Themethod of claim 40, wherein the pathogen is a virus.
 42. The method ofclaim 41, wherein the virus is from Family Togaviridae, Flaviviridae,Poxviridae or Orthomyxoviridae.
 43. The method of claim 41, wherein thevirus is from Family: Togaveridae, Genus: Alphavirus), Family:Flaviviridae, Genus: Flavivirus), Family: Poxviridae, GenusOrthopoxvirus, or Family: Orthomyxoviridae, Genus: Influenzavirus. 44.The method of claim 43, wherein the virus is chikungunya virus (CHIKV,Family: Togaviridae, Genus: Alphavirus), dengue virus serotypes 1-4 andyellow fever virus (DENY 1-4, YFV, Flaviviridae, Genus: Flavivirus),vaccinia virus (VV, Family: Poxviridae, Genus: Orthopoxvirus) orinfluenza virus (Family: Orthomyxoviridae, Genus: Infuenzavirus.
 45. Themethod of claim 40, wherein the pathogen is a bacterium.
 46. The methodof claim 45, wherein the bacterium is Campylobacter.
 47. The method ofclaim 46, wherein the Campylobacter is C. coli or C. jejuni.
 48. Themethod of claim 45, wherein the bacterium is Shigella spp.
 49. Themethod of claim 45, wherein the bacterium is Listeria spp.
 50. Themethod of claim 35, wherein the pathogen is isolated or purified priorto contacting with the Fenton reagent.
 51. The method of claim 35,wherein the methisazone reagent comprises a compound having formula I:

wherein R₁ is independently H or lower alkyl (e.g., C1-C4 alkyl)optionally substituted with —OH; wherein R₂ is independently H. loweralkyl (e.g., C1-C2 alkyl) optionally substituted with —OH or with aryl;and wherein X is independently H or halogen; and pharmaceuticallyacceptable salts thereof.
 52. The method of claim 51, wherein X and R₂are H; and wherein R₁ is H (isatin β-thiosemicarbazone), —CH₃(N-methyl-isatin β-thiosemicarbazone (methisazone)), or propyl(N-propyl-isatin β-thiosemicarbazone).
 53. The method of claim 52,wherein R₁ is —CH₃ (N-methyl-isatin β-thiosemicarbazone methisazone)).54. The method of claim 35, wherein the methisazone reagent comprisesone or more compounds each having one of formulas II-V:

wherein R₁ is H or lower alkyl (e.g., C1-C4) alkyl optionallysubstituted with —OH; and wherein X is independently H or halogen; andsalts, including pharmaceutically acceptable salts thereof;

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl) optionallysubstituted with —OH; wherein X is independently H or halogen; andwherein R₂ is independently lower alkyl (e.g., C1-C2 alkyl) optionallysubstituted with —OH, or with aryl; and salts, includingpharmaceutically acceptable salts thereof; and

wherein R₂ and R₃ are independently H, lower alkyl (e.g., C1-C2 alkyl)optionally substituted with 13 OH, or with aryl; and salts, includingpharmaceutically acceptable salts thereof and combinations thereof. 55.The method of claim 54, wherein X of formula II is H, and R₁ of formula(II) is H (isatin), —CH; (N-methyl-isatin), or propyl (N-propyl-isatin);wherein X, R₁ and R₂ of formula (III) are H (indole, 2,3-dione,3-hydrazone); wherein R₂ and R₃ of formula (IV) are H(thiosemicarbazide); and wherein R₂ and R₃ of formula (V) are H(semicarbazide).
 56. The method of claim 54, wherein the methisazonereagent comprises thiosemicarbazide and a compound having formula VI:

wherein R₁ is H or lower alkyl (e.g., C1-C4 alkyl).
 57. The method ofclaim 56, wherein R₁ is H (isatin), —CH₃ (N-methyl-isatin), or propyl(N-propyl-isatin).
 58. The method of claim 56, wherein R₁ is H (isatin).